Methods of conditioning patients for T cell therapy

ABSTRACT

The invention provides methods of increasing the efficacy of a T cell therapy in a patient in need thereof. The invention includes a method of conditioning a patient prior to a T cell therapy, wherein the conditioning involves administering a combination of cyclophosphamide and fludarabine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/262,143 filed Dec. 2, 2015, and 62/167,750 filed May 28, 2015.All of the above listed applications are incorporated herein byreference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made in the performance of a Cooperative Research andDevelopment Agreement with the National Cancer Institute (NCI), anAgency of the Department of Health and Human Services. The Government ofthe United States has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to methods of pre-conditioning a patient in needof a tumor treatment, e.g., a T cell therapy. In particular, theinvention relates to a method of improving the efficacy of a T celltherapy, including an engineered CAR T cell therapy, by firstadministering to a patient in need of the T cell therapy a conditioningchemotherapy regimen comprising cyclophosphamide and fludarabine.

BACKGROUND OF THE INVENTION

Human cancers are by their nature comprised of normal cells that haveundergone a genetic or epigenetic conversion to become abnormal cancercells. In doing so, cancer cells begin to express proteins and otherantigens that are distinct from those expressed by normal cells. Theseaberrant tumor antigens can be used by the body's innate immune systemto specifically target and kill cancer cells. However, cancer cellsemploy various mechanisms to prevent immune cells, such as T and Blymphocytes, from successfully targeting cancer cells.

Human T cell therapies rely on enriched or modified human T cells totarget and kill cancer cells in a patient. Various technologies havebeen developed to enrich the concentration of naturally occurring Tcells capable of targeting a tumor antigen or genetically modifying Tcells to specifically target a known cancer antigen. These therapieshave proven to have modest, though promising, effects on tumor size andpatient survival. However, it has proven difficult to predict whether agiven T cell therapy will be effective in each patient.

Cyclophosphamide can be administered alone or in combination with otheragents, including carmustine (BCNU) and etoposide (VP-16). As amonotherapy, cyclophosphamide can be administered by IV at 40-50 mg/kg(1.5-1.8 g/m²) as 10 to 20 mg/kg/day for 2-5 days.

Recent studies have shown that preconditioning a patient with one ormore immunosuppressive chemotherapy drugs prior to T cell infusion canincrease the effectiveness of the transplanted T cells. Rosenberg etal., Clin. Cancer. Res. (2011). However, current methods rely on highdoses of toxic and non-specific drugs, which cause painful and sometimesdeadly adverse events. As a result, there remains a need to identify aneffective preconditioning regimen for improved T cell therapies.

SUMMARY OF THE INVENTION

The present disclosure provides a method of conditioning a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide between 200 mg/m²/day and 2000 mg/m²/day and a doseof fludarabine between 20 mg/m²/day and 900 mg/m²/day.

The present disclosure further provides a method of reducing endogenouslymphocytes in a patient in need of a T cell therapy comprisingadministering to the patient a dose of cyclophosphamide between 200mg/m²/day and 2000 mg/m²/day and a dose of fludarabine between 20mg/m²/day and 900 mg/m²/day.

The present disclosure also provides a method of increasing a serumlevel of a homeostatic cytokine in a patient in need of a T cell therapycomprising administering to the patient a dose of cyclophosphamidebetween 200 mg/m²/day and 2000 mg/m²/day and a dose of fludarabinebetween 20 mg/m²/day and 900 mg/m²/day.

In certain embodiments, the homeostatic cytokine comprises interleukin 7(IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5(IL-5), gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), monocytechemotactic protein 1 (MCP-1), placental growth factor (PLGF),C-reactive protein (CRP), soluble intercellular adhesion molecule 1(sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or anycombination thereof.

The present disclosure also provides a method of enhancing an effectorfunction of administered T cells in a patient in need of a T celltherapy comprising administering to the patient a dose ofcyclophosphamide between 200 mg/m²/day and 2000 mg/m²/day and a dose offludarabine between 20 mg/m²/day and 900 mg/m²/day.

The present disclosure also provides a method of enhancing antigenpresenting cell activation and/or availability in a patient in need of aT cell therapy comprising administering to the patient a dose ofcyclophosphamide between 200 mg/m²/day and 2000 mg/m²/day and a dose offludarabine between 20 mg/m²/day and 900 mg/m²/day.

In certain embodiments, the T cell therapy is selected fromtumor-infiltrating lymphocyte (TIL) immunotherapy, autologous celltherapy, engineered autologous cell therapy (eACT), and allogeneic Tcell transplantation.

The present disclosure also provides a method of treating a patienthaving a lymphoma comprising administering daily to the patient about500 mg/m²/day of cyclophosphamide and about 60 mg/m²/day of fludarabinefor three days prior to administration of a therapeutically effectiveamount of engineered CAR T cells to the patient, wherein the engineeredCAR T cells express a chimeric antigen receptor that binds to CD19 andfurther comprises a CD28 costimulatory domain and a CD3-zeta signalingregion.

The present disclosure also provides a method of treating a patienthaving a lymphoma comprising (i) administering to the patient about 200mg/m²/day of cyclophosphamide and about 20 mg/m²/day of fludarabine and(ii) administering to the patient a therapeutically effective amount ofengineered CAR T cells, wherein the engineered CAR T cells express achimeric antigen receptor that binds to CD19 and further comprises aCD28 costimulatory domain and a CD3-zeta signaling region.

The present disclosure also provides a method of treating a patienthaving a lymphoma comprising (i) administering to the patient about 300mg/m²/day of cyclophosphamide and about 30 mg/m²/day of fludarabine and(ii) administering to the patient a therapeutically effective amount ofengineered CAR T cells, wherein the engineered CAR T cells express achimeric antigen receptor that binds to CD19 and further comprises aCD28 costimulatory domain and a CD3-zeta signaling region.

The present disclosure also provides a method of treating a patienthaving a lymphoma comprising (i) administering to the patient about 300mg/m²/day of cyclophosphamide and about 60 mg/m²/day of fludarabine and(ii) administering to the patient a therapeutically effective amount ofengineered CAR T cells, wherein the engineered CAR T cells express achimeric antigen receptor that binds to CD19 and further comprises aCD28 costimulatory domain and a CD3-zeta signaling region.

The present disclosure also provides a method of treating a patienthaving a lymphoma comprising (i) administering to the patient about 500mg/m²/day of cyclophosphamide and about 60 mg/m²/day of fludarabine and(ii) administering to the patient a therapeutically effective amount ofengineered CAR T cells, wherein the engineered CAR T cells express achimeric antigen receptor that binds to CD19 and further comprises aCD28 costimulatory domain and a CD3-zeta signaling region.

The present disclosure also provides a method of treating a patienthaving a lymphoma comprising administering to the patient atherapeutically effective amount of engineered CAR T cells, wherein thepatient has been conditioned by administration of about 500 mg/m²/day ofcyclophosphamide and about 60 mg/m²/day of fludarabine and wherein theengineered CAR T cells express a chimeric antigen receptor that binds toCD19 and further comprises a CD28 costimulatory domain and a CD3-zetasignaling region.

The present disclosure also provides a kit comprising (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at a dose between 200 mg/m²/day and 2000 mg/m²/day andfludarabine at a dose between 20 mg/m²/day and 900 mg/m²/day daily forthree days to a patient in need of an engineered CAR T cell therapyprior to the therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of an example CAR-engineered Tcell and its construction. In this exemplary CAR-engineered T cell, thetarget binding domain comprises an antibody derived scFv domain, thecostimulatory domain is derived from CD28, and the essential activatingdomain is derived from CD3ζ (zeta). A CAR vector construct can becarried by a viral vector and then incorporated into a T cell genome.The CAR construct can then be expressed by the T cell as a transmembraneprotein.

FIGS. 2A and 2B show patient disease responses following treatment withanti-CD-19 CAR+ T cells. The best responses of patients with B cellmalignancies are shown in FIG. 2A as a percent change in diseasecondition. Dashed bars indicate a complete response (CR). Shaded barsindicate a partial response. White bars indicate a stable disease (SD).Black bars indicate progressive disease (PD). FIG. 2B shows patientdisease responses relative to months post-CAR+ T cell infusion. Solidblack bars indicate partial response (PR), and grey bars indicatecomplete response (CR). Breaks in the bars marked with “PD” indicatethat the patient experienced a progressive disease. Inverted trianglesmark the time of T cell infusion. Solid circles indicate the time of Bcell recovery. White circles indicate the time of CAR+ T cell clearancefrom the patient's blood. A horizontal arrow indicates that thepatient's response is ongoing.

FIG. 3 provides a sample diagram of a phase 1 clinical trial directed todetermining the safety, efficacy, and dose limiting toxicities oftreating a patient with 500 mg/m²/day cyclophosphamide, 30 mg/m²/dayfludarabine, and 2×10⁶ anti-CD19 CAR+ T cells/kg.

FIGS. 4A-4H shows serum levels of selected cytokine analytes before andafter conditioning with 300 mg/m²/day cyclophosphamide and 30 mg/m²/dayfludarabine. The serum levels of interleukin 15 (IL-15; FIG. 4A),monocyte chemotactic protein 1 (MCP-1; FIG. 4B), gamma-induced protein10 (IP-10; FIG. 4C), placental growth factor (PLGF; FIG. 4D), solubleintercellular adhesion molecule 1 (sICAM-1; FIG. 4E), C-reactive protein(CRP; FIG. 4F), vascular endothelial growth factor D (VEGF-D; FIG. 4G),and macrophage inflammatory protein 1β (MIP-1b; FIG. 4H) are shownpre-administration and post-administration of 300 mg/m² cyclophosphamideand 30 mg/m² fludarabine. Pre-administration serum was collected betweenday −12 and day −5, and post-administration serum was collected on day 0prior to T cell therapy administration (FIGS. 4A-4H).

FIGS. 5A-H show the fold change in the serum levels of select cytokineanalytes following conditioning with 300 mg/m²/day cyclophosphamide and30 mg/m²/day fludarabine in patients who either responded or did notrespond to subsequence T cell therapy. The fold change in the serumlevels of IL-15 (FIG. 5A), MCP-1 (FIG. 5B), IP-10 (FIG. 5C), PLGF (FIG.5D), sICAM-1 (FIG. 5E), CRP (FIG. 5F), VEGF (FIG. 5G), and MIP-1b (FIG.5H) are shown for responders and non-responders. Horizontal linesindicate the average (FIGS. 5A-H). Individual patient IL-15 changes areshown in FIG. 5A, and each patient's disease responsiveness is indicatednext to each data point as a partial response (PR), complete response(CR), stable disease (SD), or progressive disease (PD).

FIGS. 6A-6V show the serum concentration of select cytokine analytesmeasured at various time points from day −10 to day 18 for patientsadministered 300 mg/m²/day cyclophosphamide and 30 mg/m²/day fludarabineprior to receiving a T cell therapy on day 0. The serum concentration ofgranulocyte macrophage colony-stimulating factor (GM-CSF; FIG. 6A), IL-2(FIG. 6B), MCP-1 (FIG. 6C), IL-6 (FIG. 6D), IL-10 (FIG. 6E), MCP-4 (FIG.6F), CRP (FIG. 6G), interferon gamma (IFNγ; FIG. 6H), granzyme A (FIG.6I), IL-15 (FIG. 6J), IL-5 (FIG. 6K), granzyme B (FIG. 6L), IL-8 (FIG.6M), IP-10 (FIG. 6N), MIP-1b (FIG. 6O), PLGF (FIG. 6P), IL-16 (FIG. 6Q),thymus and activation regulated chemokine (TARC; FIG. 6R), eotaxin-3(FIG. 6S), sICAM-1 (FIG. 6T), soluble vascular adhesion molecule 1(sVCAM; FIG. 6U), and (SAA; FIG. 6V) are shown.

FIGS. 7A-7I show the serum concentration of selected cytokine analytesmeasured pre- and post-administration of 300 mg/m²/day cyclophosphamideand 30 mg/m²/day fludarabine. Post-administration sera were collectedright before T cell infusion. The serum concentrations of IL-15 (FIG.7A), IL-7 (FIG. 7B), PLGF (FIG. 7C), CRP (FIG. 7D), IL-5 (FIG. 7E),IL-10 (FIG. 7F), MCP-1 (FIG. 7G), IP-10 (FIG. 7H), and sICAM-1 (FIG. 7I)are shown. Each data point represents a single patient. Horizontal barsshow the average (FIGS. 7A-7I). P value of Wilcoxon matched-pairs signedrank test was applied to analytes measured pre-conditioning andpost-conditioning, and corresponding P values are shown (FIGS. 7A-7I).Some IL-7 values were above the upper limit of quantitation (ULOQ; FIG.7B).

FIG. 8A-8L shows the in vitro production of various cytokine analytesproduced by anti-CD19 CAR+ T cells (K562-CD19) as compared to a negativecontrol (K562-NGFR) following stimulation with K562 cells. Theconcentrations of GM-CSF (FIG. 8A), IL-2 (FIG. 8B), IFNγ (FIG. 8C), IL-5(FIG. 8D), IL-4 (FIG. 8E), IL-13 (FIG. 8F), tumor necrosis factor alpha(TNFα; FIG. 8G), IL-6 (FIG. 8H), granzyme B (FIG. 8I), MIP-1β (FIG. 8J),MIP-1α (FIG. 8K), and soluble CD137 (FIG. 8L) are shown for control andanti-CD19 CAR+ T cells. T1, T2, and immune homeostatic cytokines (FIGS.8A-8F) and pro-inflammatory cytokines and chemokines (FIGS. 8G-8L) arelabeled accordingly. Data was collected pre-infusion by co-incubatingproduct T cells with K562-CD19 or control K562-NGFR cells and measuringthe concentration of the listed analytes in the medium (FIGS. 8A-8L).

FIG. 9A-9C shows the percent of anti-CD19 CAR+ T cells (K562-CD19)expressing various cytokines following engagement with a target antigenas compared to a negative control (K562-NGFR). The percent of cellsexpressing CD107α (FIG. 9A), 4-1BB (FIG. 9B), and programmed death 1(PD-1; FIG. 9C) are shown. Data was collected pre-infusion byco-incubating product T cells with K562-CD19 or control K562-NGFR cellsand measuring the concentration of the select activating markers in themedium (FIGS. 9A-9C). P values shown indicate the results of a paired Ttest comparing K562-CD19 test cells with K562-NGFR negative controlcells (FIGS. 9A-9C).

FIG. 10 illustrates the various characteristics of the product T cellsand peripheral blood lymphocytes (PBLs) in view of the manufacturingtime (days). The data include the percent of anti-CD-19 CAR+ T cellsdetected in the product versus the PBL; the ratio of CD8 to CD4 in theproduct versus the PBL; the relative occurrence of naïve, central memory(Tcm), effector memory (Tem), and effector (Teff) T cells within theanti-CD19 CAR+ CD8+ T cell population; and the relative occurrence ofnaïve, central memory (Tcm), effector memory (Tem), and effector (Teff)T cells within the anti-CD19 CAR+CD4+ T cell population (FIG. 10). Thephenotypic analysis of product T cells before infusion and of PBL duringpeak expansion in blood was done on anti-CD19 CAR+ T cells (FIG. 10).The p-value represents the results of a rank test of association betweenmanufacturing time and T cell subset composition.

FIG. 11 shows expression profile of cytokines, chemokines and othermarkers observed following NHL patient conditioning according to theinvention. CRP: C reactive protein. PLGF: Placental growth factor.MCP-1: Monocyte chemoattractant protein-1.

FIG. 12 sets forth quantification of changes observed in cytokines,chemokines and other markers following Conditioning withCyclophosphamide and Fludarabine according to the invention.

FIG. 13 shows the magnitude of change in circulating IL-15 and perforinfollowing conditioning chemotherapy associated with objective response.P values were not adjusted for multiplicity. Analysis executed onmarkers measured prior to CAR T cell infusion.

FIG. 14 sets forth a biomarker analysis of cytokines, chemokines, andeffector molecules. Markers were ordered within each category ofbiomarkers by low to high p-value using Wilcoxon signed-rank test. Thosemodified in a majority of patients and with p values of <0.05 werepresented. Only 7 out of 41 measured markers showed changes in amajority of patients, associated with p<0.05. Analysis was executed onmarkers measured prior to CAR T cell infusion.

FIGS. 15A-15H set forth sequential induction and clearance of immunehomeostatic, inflammatory, and modulating cytokines, chemokines andimmune effector molecules. Representative markers are shown. A total of22 out of 41 measured markers showed an elevation post CAR T-celltreatment in at least 50% of the patients, at least 2-fold higher thanbaseline values: IL-15, IL-7, IL-2, Granzyme B, Granzyme A, CRP, IL-6,GM-CSF, IL-5, IFNg, IL-10, MCP-1, MCP-4, IP-10, IL-8, TARC, MIP1a,MIP1b, PLGF, VEGF-D, sICAM-1 and FGF-2. Peaking observed on days 3-4 forimmune homeostatic cytokines & chemokines.

FIGS. 16A-16H set forth the sequential induction and clearance of immunehomeostatic, inflammatory, and modulating cytokines, chemokines andimmune effector molecules. Representative markers are shown. A total of22 out of 41 measured markers showed elevation post CAR T cell treatmentin at least 50% of the patients, at least 2-fold higher than baselinevalues: IL-15, IL-7, IL-2, Granzyme B, Granzyme A, CRP, IL-6, GM-CSF,IL-5, IFNg, IL-10, MCP-1, MCP-4, IP-10, IL-8, TARC, MIP1a, MIP1b, PLGF,VEGF-D, sICAM-1 and FGF-2. Peaking was observed on days 5-7 for immunemodulating cytokines and chemokines. “ULOQ”: upper limit ofquantitation.

FIG. 17 shows the change in treatment-related biomarkers and clinicalresponse induced by anti-CD19 CAR T cells according to the invention.Maximum fold change of marker levels post-CAR T cell treatment versusbaseline (pre-conditioning). Each line represents an individual subject.The Wilcoxon rank-sum test was used to compare the maximum fold changevalues across responder vs non-responder groups, for all 41 biomarkersevaluated. P-values were not adjusted for multiplicity, and only thosebiomarkers with p<0.10 were shown: p values for IL-7 and sICAM-1 were<0.05. The association was also applicable to changes in absolute levelsof IL-7 (p=0.0165), IL-15 (p=0.0314) and IL-15 (p=0.041).

FIGS. 18A-18G show the change in the level of analytes before and afterconditioning with cyclophosphamide and fludarabine. FIGS. 18A-18F showthe pre and post levels of IL-15 (FIG. 18A), IP-10 (FIG. 18B), CRP (FIG.18C), IL-7 (FIG. 18D), MCP-1 (FIG. 18E), and perforin (FIG. 18F). FIG.18G summarizes the change in serum levels of various analytes and thecorresponding p values.

FIG. 19A-19D shows the correlation between change in analyte level afterconditioning and the objective response to CAR T cell therapy for IL-15(FIG. 19A), IP-10 (FIG. 19B), and perforin (FIG. 19C). FIG. 19D providesa summary of the statistical significance of the data provided in eachof FIGS. 19A-19C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of conditioning a patient inneed of a T cell therapy, e.g., an engineered CAR T cell therapy, e.g.,an autologous cell therapy (eACT™), comprising administeringcyclophosphamide and fludarabine prior to administering the T celltherapy. Pre-conditioning patients prior to T cell therapies with thesedoses of cyclophosphamide and fludarabine improves the efficacy of the Tcell therapy by reducing the number of endogenous lymphocytes andincreasing the serum level of homeostatic cytokines and/or pro-immunefactors present in the patient. This creates a more optimalmicroenvironment for the transplanted T cells to proliferate onceadministered to the patient. Pre-conditioning at the doses describedherein surprisingly reduced the number of endogenous lymphocytes whileminimizing toxicity associated with cyclophosphamide and fludarabinetreatment. The invention is directed to decreasing the cyclophosphamideand fludarabine doses for preconditioning prior to a T cell therapy.Administration of the specific doses of cyclophosphamide and fludarabineinduces the optimal level of cytokine availability for transferred Tcells, while providing lower toxicities overall to the patient subjectto a T cell therapy.

Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their System Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects of the disclosure, which can be hadby reference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification in its entirety.

The term “activation” refers to the state of an immune cell, e.g., a Tcell, that has been sufficiently stimulated to induce detectablecellular proliferation. Activation can also be associated with inducedcytokine production and detectable effector functions. The term“activated T cells” refers to, among other things, T cells that areundergoing cell division.

“Administering” refers to the physical introduction of an agent to asubject, using any of the various methods and delivery systems known tothose skilled in the art. Exemplary routes of administration for theformulations disclosed herein include intravenous, intramuscular,subcutaneous, intraperitoneal, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion, as well as in vivoelectroporation. In some embodiments, the formulation is administeredvia a non-parenteral route, e.g., orally. Other non-parenteral routesinclude a topical, epidermal or mucosal route of administration, forexample, intranasally, vaginally, rectally, sublingually or topically.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended or undesirable sign (including an abnormal laboratoryfinding), symptom, medical occurrence, or disease associated with theuse of a medical treatment. The definition of adverse events includesworsening of a pre-existing medical condition. Worsening indicates thata pre-existing medical condition has increased in severity, frequency,and/or duration or has an association with a worse outcome.

The term “antibody” (Ab) includes, without limitation, a glycoproteinimmunoglobulin which binds specifically to an antigen. In general, andantibody can comprise at least two heavy (H) chains and two light (L)chains interconnected by disulfide bonds, or an antigen-binding portionthereof. Each H chain comprises a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. The heavychain constant region comprises three constant domains, CH1, CH2 andCH3. Each light chain comprises a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprises one constant domain, CL. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL comprises three CDRs and four FRs, arranged from amino-terminusto carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe Abs may mediate the binding of the immunoglobulin to host tissues orfactors, including various cells of the immune system (e.g., effectorcells) and the first component (C1q) of the classical complement system.

An immunoglobulin may derive from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. IgGsubclasses are also well known to those in the art and include but arenot limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to theAb class or subclass (e.g., IgM or IgG1) that is encoded by the heavychain constant region genes. The term “antibody” includes, by way ofexample, both naturally occurring and non-naturally occurring Abs;monoclonal and polyclonal Abs; chimeric and humanized Abs; human ornonhuman Abs; wholly synthetic Abs; and single chain Abs. A nonhuman Abmay be humanized by recombinant methods to reduce its immunogenicity inman. Where not expressly stated, and unless the context indicatesotherwise, the term “antibody” also includes an antigen-binding fragmentor an antigen-binding portion of any of the aforementionedimmunoglobulins, and includes a monovalent and a divalent fragment orportion, and a single chain Ab.

An “antigen binding molecule” or “antibody fragment” refers to anyportion of an antibody less than the whole. An antigen binding moleculecan include the antigenic complementarity determining regions (CDRs).Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)2, and Fv fragments, dAb, linear antibodies, scFvantibodies, and multispecific antibodies formed from antigen bindingmolecules.

An “antigen” refers to any molecule that provokes an immune response oris capable of being bound by an antibody. The immune response mayinvolve either antibody production, or the activation of specificimmunologically-competent cells, or both. A person of skill in the artwould readily understand that any macromolecule, including virtually allproteins or peptides, can serve as an antigen. An antigen can beendogenously expressed, i.e. expressed by genomic DNA, or can berecombinantly expressed. An antigen can be specific to a certain tissue,such as a cancer cell, or it can be broadly expressed. In addition,fragments of larger molecules can act as antigens. In one embodiment,antigens are tumor antigens.

The term “autologous” refers to any material derived from the sameindividual to which it is later to be re-introduced. For example, theengineered autologous cell therapy (eACT™) method described hereininvolves collection of lymphocytes from a patient, which are thenengineered to express, e.g., a CAR construct, and then administered backto the same patient.

The term “allogeneic” refers to any material derived from one individualwhich is then introduced to another individual of the same species,e.g., allogeneic T cell transplantation.

A “cancer” refers to a broad group of various diseases characterized bythe uncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth results in the formation of malignant tumors thatinvade neighboring tissues and may also metastasize to distant parts ofthe body through the lymphatic system or bloodstream. A “cancer” or“cancer tissue” can include a tumor. Examples of cancers that can betreated by the methods of the present invention include, but are notlimited to, cancers of the immune system including lymphoma, leukemia,and other leukocyte malignancies. In some embodiments, the methods ofthe present invention can be used to reduce the tumor size of a tumorderived from, for example, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, testicular cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B celllymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicularlymphoma (FL), transformed follicular lymphoma, splenic marginal zonelymphoma (SMZL), cancer of the esophagus, cancer of the small intestine,cancer of the endocrine system, cancer of the thyroid gland, cancer ofthe parathyroid gland, cancer of the adrenal gland, sarcoma of softtissue, cancer of the urethra, cancer of the penis, chronic or acuteleukemia, acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia (ALL) (including non T cell ALL), chroniclymphocytic leukemia (CLL), solid tumors of childhood, lymphocyticlymphoma, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor,brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer, squamous cell cancer, T-cell lymphoma, environmentally inducedcancers including those induced by asbestos, other B cell malignancies,and combinations of said cancers. The particular cancer can beresponsive to chemo- or radiation therapy or the cancer can berefractory. A refractor cancer refers to a cancer that is not amendableto surgical intervention and the cancer is either initially unresponsiveto chemo- or radiation therapy or the cancer becomes unresponsive overtime.

An “anti-tumor effect” as used herein, refers to a biological effectthat can present as a decrease in tumor volume, a decrease in the numberof tumor cells, a decrease in tumor cell proliferation, a decrease inthe number of metastases, an increase in overall or progression-freesurvival, an increase in life expectancy, or amelioration of variousphysiological symptoms associated with the tumor. An anti-tumor effectcan also refer to the prevention of the occurrence of a tumor, e.g., avaccine.

The term “progression-free survival,” which can be abbreviated as PFS,as used herein refers to the time from the treatment date to the date ofdisease progression per the revised IWG Response Criteria for MalignantLymphoma or death from any cause.

“Disease progression” is assessed by measurement of malignant lesions onradiographs or other methods should not be reported as adverse events.Death due to disease progression in the absence of signs and symptomsshould be reported as the primary tumor type (e.g., DLBCL).

The “duration of response,” which can be abbreviated as DOR, as usedherein refers to the period of time between a subject's first objectiveresponse to the date of confirmed disease progression, per the revisedIWG Response Criteria for Malignant Lymphoma, or death.

The term “overall survival,” which can be abbreviated as OS, is definedas the time from the date of treatment to the date of death.

A “cytokine,” as used herein, refers to a non-antibody protein that isreleased by one cell in response to contact with a specific antigen,wherein the cytokine interacts with a second cell to mediate a responsein the second cell. A cytokine can be endogenously expressed by a cellor administered to a subject. Cytokines may be released by immune cells,including macrophages, B cells, T cells, and mast cells to propagate animmune response. Cytokines can induce various responses in the recipientcell. Cytokines can include homeostatic cytokines, chemokines,pro-inflammatory cytokines, effectors, and acute-phase proteins. Forexample, homeostatic cytokines, including interleukin (IL) 7 and IL-15,promote immune cell survival and proliferation, and pro-inflammatorycytokines can promote an inflammatory response. Examples of homeostaticcytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7,IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examplesof pro-inflammatory cytokines include, but are not limited to, IL-1a,IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta,fibroblast growth factor (FGF) 2, granulocyte macrophagecolony-stimulating factor (GM-CSF), soluble intercellular adhesionmolecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1),vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placentalgrowth factor (PLGF). Examples of effectors include, but are not limitedto, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin.Examples of acute phase-proteins include, but are not limited to,C-reactive protein (CRP) and serum amyloid A (SAA).

“Chemokines” are a type of cytokine that mediates cell chemotaxis, ordirectional movement. Examples of chemokines include, but are notlimited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derivedchemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 orCCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a),MIP-1β (MIP-1b), gamma-induced protein 10 (IP-10), and thymus andactivation regulated chemokine (TARC or CCL17).

Other examples of analytes and cytokines of the present inventioninclude, but are not limited to chemokine (C-C motif) ligand (CCL) 1,CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocytechemoattractant protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9,IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, granulocyte colony-stimulatingfactor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM),CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), aproliferation-inducing ligand (APRIL), CD70, CD153, CD178,glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosisfactor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-relatedleukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducingligand (TRAIL).

The terms “serum level” and “serum concentration” are usedinterchangeably as used herein and refer to the amount of an analyte inthe serum of a subject. Serum levels of a given analyte can be measuredusing any method known in the art. For example, cytokine serum levelscan be measured using an enzyme-linked immunosorbent assay (ELISA). Inone particular embodiment, cytokine serum levels can be measured usingan EMDmillipore LUMINEX® xMAP® multiplex assay.

“Dosing interval,” as used herein, means the amount of time that elapsesbetween multiple doses of a formulation disclosed herein beingadministered to a subject. Dosing interval can thus be indicated asranges.

Doses described herein can be presented as a “weight based dose” or as a“body surface area (BSA) based dose.” A weight based dose is a dose thatis administered to a patient that is calculated based on the weight ofthe patient, e.g., mg/kg. A BSA based dose is a dose that isadministered to a patient that is calculated based on the surface areaof the patient, e.g., mg/m². The two forms of dose measurement can beconverted for human dosing by multiplying the weight based dose by 37 ordividing the BSA based dose by 37. For example, a dose of 60 mg/kg to beadministered to a human subject is equivalent to a 2220 mg/m² dose ofthe same drug to be administered to the same subject.

The term “dosing frequency” as used herein refers to the frequency ofadministering doses of a formulation disclosed herein in a given time.Dosing frequency can be indicated as the number of doses per a giventime. For example, cyclophosphamide can be administered as a single doseper day on each of 5 consecutive days, as a single dose per day on eachof 4 consecutive days, as a single dose per day on each of 3 consecutivedays, as a single dose per day on each of 2 consecutive days, or as asingle dose on 1 day. In certain embodiments, the cyclophosphamide isadministered as 1 dose per day for 3 consecutive days or 1 dose per dayfor 2 consecutive days. Fludarabine can be administered as a single doseper day on each of 8 consecutive days, as a single dose per day on eachof 7 consecutive days, as a single dose per day on each of 6 consecutivedays, as a single dose per day on each of 5 consecutive days, as asingle dose per day on each of 4 consecutive days, as a single dose perday on each of 3 consecutive days, as a single dose per day on each of 2consecutive days, or as a single dose on 1 day. In other embodiments,the fludarabine is administered as 1 dose per day for 5 consecutive daysor as 1 dose per day for 3 consecutive days.

A “therapeutically effective amount,” “effective dose,” “effectiveamount,” or “therapeutically effective dosage” of a drug or therapeuticagent is any amount of the drug that, when used alone or in combinationwith another therapeutic agent, protects a subject against the onset ofa disease or promotes disease regression evidenced by a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction. The ability of a therapeuticagent to promote disease regression can be evaluated using a variety ofmethods known to the skilled practitioner, such as in human subjectsduring clinical trials, in animal model systems predictive of efficacyin humans, or by assaying the activity of the agent in in vitro assays.

The term “lymphocyte” as used herein includes natural killer (NK) cells,T cells, or B cells. NK cells are a type of cytotoxic (cell toxic)lymphocyte that represent a major component of the inherent immunesystem. NK cells reject tumors and cells infected by viruses. It worksthrough the process of apoptosis or programmed cell death. They weretermed “natural killers” because they do not require activation in orderto kill cells. T-cells play a major role in cell-mediated-immunity (noantibody involvement). Its T-cell receptors (TCR) differentiatethemselves from other lymphocyte types. The thymus, a specialized organof the immune system, is primarily responsible for the T cell'smaturation. There are six types of T-cells, namely: Helper T-cells(e.g., CD4+ cells), Cytotoxic T-cells (also known as TC, cytotoxic Tlymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cells or killerT cell), Memory T-cells ((i) stem memory T_(SCM) cells, like naivecells, are CD45RO−, CCR7+, CD45RA+, CD62L+(L-selectin), CD27+, CD28+ andIL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, andLFA-1, and show numerous functional attributes distinctive of memorycells); (ii) central memory T_(CM) cells express L-selectin and theCCR7, they secrete IL-2, but not IFNγ or IL-4, and (iii) effector memoryT_(EM) cells, however, do not express L-selectin or CCR7 but produceeffector cytokines like IFNγ and IL-4), Regulatory T-cells (Tregs,suppressor T cells, or CD4+CD25+ regulatory T cells), Natural KillerT-cells (NKT) and Gamma Delta T-cells. B-cells, on the other hand, playa principal role in humoral immunity (with antibody involvement). Itmakes antibodies and antigens and performs the role ofantigen-presenting cells (APCs) and turns into memory B-cells afteractivation by antigen interaction. In mammals, immature B-cells areformed in the bone marrow, where its name is derived from.

The term “genetically engineered” or “engineered” refers to a method ofmodifying the genome of a cell, including, but not limited to, deletinga coding or non-coding region or a portion thereof or inserting a codingregion or a portion thereof. In some embodiments, the cell that ismodified is a lymphocyte, e.g., a T cell, which can either be obtainedfrom a patient or a donor. The cell can be modified to express anexogenous construct, such as, e.g., a chimeric antigen receptor (CAR) ora T cell receptor (TCR), which is incorporated into the cell's genome.

An “immune response” refers to the action of a cell of the immune system(for example, T lymphocytes, B lymphocytes, natural killer (NK) cells,macrophages, eosinophils, mast cells, dendritic cells and neutrophils)and soluble macromolecules produced by any of these cells or the liver(including Abs, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom a vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues.

The term “immunotherapy” refers to the treatment of a subject afflictedwith, or at risk of contracting or suffering a recurrence of, a diseaseby a method comprising inducing, enhancing, suppressing or otherwisemodifying an immune response. Examples of immunotherapy include, but arenot limited to, T cell therapies. T cell therapy can include adoptive Tcell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy,autologous cell therapy, engineered autologous cell therapy (eACT), andallogeneic T cell transplantation. However, one of skill in the artwould recognize that the conditioning methods disclosed herein wouldenhance the effectiveness of any transplanted T cell therapy. Examplesof T cell therapies are described in U.S. Patent Publication Nos.2014/0154228 and 2002/0006409, U.S. Pat. No. 5,728,388, andInternational Publication No. WO 2008/081035.

The T cells of the immunotherapy can come from any source known in theart. For example, T cells can be differentiated in vitro from ahematopoietic stem cell population, or T cells can be obtained from asubject. T cells can be obtained from, e.g., peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In addition, the T cells can be derived fromone or more T cell lines available in the art. T cells can also beobtained from a unit of blood collected from a subject using any numberof techniques known to the skilled artisan, such as FICOLL™ separationand/or apheresis. Additional methods of isolating T cells for a T celltherapy are disclosed in U.S. Patent Publication No. 2013/0287748, whichis herein incorporated by references in its entirety.

The term “engineered Autologous Cell Therapy,” which can be abbreviatedas “eACT™,” also known as adoptive cell transfer, is a process by whicha patient's own T cells are collected and subsequently geneticallyaltered to recognize and target one or more antigens expressed on thecell surface of one or more specific tumor cells or malignancies. Tcells can be engineered to express, for example, chimeric antigenreceptors (CAR) or T cell receptor (TCR). CAR positive (+) T cells areengineered to express an extracellular single chain variable fragment(scFv) with specificity for a particular tumor antigen linked to anintracellular signaling part comprising a costimulatory domain and anactivating domain. The costimulatory domain can be derived from, e.g.,CD28, and the activating domain can be derived from, e.g., CD3-zeta(FIG. 1). In certain embodiments, the CAR is designed to have two,three, four, or more costimulatory domains. The CAR scFv can be designedto target, for example, CD19, which is a transmembrane protein expressedby cells in the B cell lineage, including all normal B cells and B cellmalignances, including but not limited to NHL, CLL, and non-T cell ALL.Example CAR+ T cell therapies and constructs are described in U.S.Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and2014/0050708, and these references are incorporated by reference intheir entirety.

A “patient” as used herein includes any human who is afflicted with acancer (e.g., a lymphoma or a leukemia). The terms “subject” and“patient” are used interchangeably herein.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

“Stimulation,” as used herein, refers to a primary response induced bybinding of a stimulatory molecule with its cognate ligand, wherein thebinding mediates a signal transduction event. A “stimulatory molecule”is a molecule on a T cell, e.g., the T cell receptor (TCR)/CD3 complex,that specifically binds with a cognate stimulatory ligand present on anantigen present cell. A “stimulatory ligand” is a ligand that whenpresent on an antigen presenting cell (e.g., an aAPC, a dendritic cell,a B-cell, and the like) can specifically bind with a stimulatorymolecule on a T cell, thereby mediating a primary response by the Tcell, including, but not limited to, activation, initiation of an immuneresponse, proliferation, and the like. Stimulatory ligands include, butare not limited to, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

A “costimulatory signal,” as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to aT cell response, such as, but not limited to, proliferation and/orupregulation or down regulation of key molecules.

A “costimulatory ligand” as used herein, includes a molecule on anantigen presenting cell that specifically binds a cognate co-stimulatorymolecule on a T cell. Binding of the costimulatory ligand provides asignal that mediates a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. Acostimulatory ligand induces a signal that is in addition to the primarysignal provided by a stimulatory molecule, for instance, by binding of aT cell receptor (TCR)/CD3 complex with a major histocompatibilitycomplex (MHC) molecule loaded with peptide. A co-stimulatory ligand caninclude, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86),programmed death (PD) L1, PD-L2, 4-1BB ligand, OX40 ligand, induciblecostimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD30 ligand, CD40, CD70, CD83, human leukocyte antigen G (HLA-G), MHCclass I chain-related protein A (MICA), MHC class I chain-relatedprotein B (MICB), herpes virus entry mediator (HVEM), lymphotoxin betareceptor, 3/TR6, immunoglobulin-like transcript (ILT) 3, ILT4, anagonist or antibody that binds Toll ligand receptor and a ligand thatspecifically binds with B7-H3. A co-stimulatory ligand includes, withoutlimitation, an antibody that specifically binds with a co-stimulatorymolecule present on a T cell, such as, but not limited to, CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, tumor necrosis factor superfamily member 14(TNFSF14 or LIGHT), natural killer cell receptor C (NKG2C), B7-H3, and aligand that specifically binds with CD83.

A “costimulatory molecule” is a cognate binding partner on a T cell thatspecifically binds with a costimulatory ligand, thereby mediating acostimulatory response by the T cell, such as, but not limited to,proliferation. Costimulatory molecules include, but are not limited to,CD27, CD28, 4-1BB, OX40, CD30, CD40, CD83, PD-1, ICOS, LFA-1, CD2, CD7,TNFSF14 (LIGHT), NKG2C, B7-H3, an MEW class 1 molecule, B- andT-lymphocyte attenuator (BTLA), and a Toll ligand receptor.

The terms “conditioning” and “pre-conditioning” are used interchangeablyherein and indicate preparing a patient in need of a T cell therapy fora suitable condition. Conditioning as used herein includes, but is notlimited to, reducing the number of endogenous lymphocytes, removing acytokine sink, increasing a serum level of one or more homeostaticcytokines or pro-inflammatory factors, enhancing an effector function ofT cells administered after the conditioning, enhancing antigenpresenting cell activation and/or availability, or any combinationthereof prior to a T cell therapy. In one embodiment, “conditioning”comprises increasing a serum level of one or more cytokines, e.g.,interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10),interleukin 5 (IL-5), gamma-induced protein 10 (IP-10), interleukin 8(IL-8), monocyte chemotactic protein 1 (MCP-1), placental growth factor(PLGF), C-reactive protein (CRP), soluble intercellular adhesionmolecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), orany combination thereof. In another embodiment, “conditioning” comprisesincreasing a serum level of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or anycombination thereof.

The terms “reducing” and “decreasing” are used interchangeably hereinand indicate any change that is less than the original. “Reducing” and“decreasing” are relative terms, requiring a comparison between pre- andpost-measurements. “Reducing” and “decreasing” include completedepletions.

“Treatment” or “treating” of a subject refers to any type ofintervention or process performed on, or the administration of an activeagent to, the subject with the objective of reversing, alleviating,ameliorating, inhibiting, slowing down or preventing the onset,progression, development, severity or recurrence of a symptom,complication or condition, or biochemical indicia associated with adisease. In one embodiment, “treatment” or “treating” includes a partialremission. In another embodiment, “treatment” or “treating” includes acomplete remission.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 10% (i.e., ±10%). For example, about 3 mg can include anynumber between 2.7 mg and 3.3 mg (for 10%). Furthermore, particularlywith respect to biological systems or processes, the terms can mean upto an order of magnitude or up to 5-fold of a value. When particularvalues or compositions are provided in the application and claims,unless otherwise stated, the meaning of “about” or “comprisingessentially of” should be assumed to be within an acceptable error rangefor that particular value or composition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

Various aspects of the invention are described in further detail in thefollowing subsections.

Methods of the Invention

The present invention is directed to methods of conditioning a patientin need of a T cell therapy, comprising administering to the patientcyclophosphamide and fludarabine. The present invention shows thatconditioning a patient with between about 200 mg/m²/day and about 2000mg/m²/day cyclophosphamide and between about 20 mg/m²/day and 900mg/m²/day fludarabine enhances the effectiveness of a T cell therapysubsequently administered to the patient, while reducing the occurrenceand/or severity of adverse events associated with higher doses ofcyclophosphamide and/or fludarabine.

The present invention identifies that administration of cyclophosphamideand fludarabine prior to administration of a T cell therapy reduces thenumber of endogenous lymphocytes. The endogenous lymphocytes that arereduced can include, but is not limited to, endogenous regulatory Tcells, B cells, natural killer cells, CD4+ T cells, CD8+ T cells, or anycombination thereof, which can inhibit the anti-tumor effect ofadoptively transferred T cells. Endogenous lymphocytes can compete withadoptively transferred T cells for access to antigens and supportivecytokines. Pretreatment with cyclophosphamide and fludarabine removesthis competition, resulting in an increase in the level of endogenouscytokines. Once the adoptively transferred T cells are administered tothe patient, they are exposed to increased levels of endogenoushomeostatic cytokines or pro-inflammatory factors. In addition,cyclophosphamide and fludarabine treatment can cause tumor cell death,leading to increased tumor antigen in the patient's serum. This canenhance antigen-presenting cell activation and or availability in thepatient, prior to receiving a T cell therapy. Not bound by any theory,conditioning with cyclophosphamide and fludarabine modifies the immuneenvironment through induction of molecules that can favor thehomeostatic expansion, activation and trafficking of T cells.

Previous studies used high doses of cyclophosphamide and fludarabine toreduce endogenous lymphocyte numbers. However, these harsh conditioningregimens are associated with serious, and potentially fatal, adverseevents. Surprisingly, the present method was found to increase theeffectiveness of adoptively transferred T cells while mitigating theoccurrence and severity of adverse events.

In some embodiments, administration of cyclophosphamide and fludarabinereduces endogenous lymphocytes. In some embodiments, administration ofcyclophosphamide and fludarabine increases the availability of ahomeostatic cytokine. In some embodiments, administration ofcyclophosphamide and fludarabine enhances an effector function of Tcells administered after the conditioning. In some embodiments,administration of cyclophosphamide and fludarabine enhances antigenpresenting cell activation and/or availability.

In one embodiment, the invention includes a method of conditioning apatient in need of a T cell therapy comprising administering to thepatient a dose of cyclophosphamide between about 200 mg/m²/day and about2000 mg/m²/day and a dose of fludarabine between about 20 mg/m²/day andabout 900 mg/m²/day. In another embodiment, the invention includes amethod of conditioning a patient in need of a T cell therapy comprisingadministering to the patient a dose of cyclophosphamide between about200 mg/m²/day and about 2000 mg/m²/day (e.g., 200 mg/m²/day, 300mg/m²/day, or 500 mg/m²/day) and a dose of fludarabine between about 20mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30mg/m²/day, or 60 mg/m²/day), wherein the patient exhibits increasedserum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP,sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and/orIL-7, or decreased serum levels of perforin and/or MIP-1b after theadministration of the cyclophosphamide and fludarabine. In oneembodiment, the invention includes a method of conditioning a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide between about 1110 mg/m²/day and about 2000mg/m²/day and a dose of fludarabine between about 20 mg/m²/day and about900 mg/m²/day, e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60mg/m²/day. In another embodiment, the invention includes a method ofconditioning a patient in need of a T cell therapy comprisingadministering to the patient a dose of cyclophosphamide between about1110 mg/m²/day and about 2000 mg/m²/day and a dose of fludarabinebetween about 20 mg/m²/day and about 900 mg/m²/day, e.g., 20 mg/m²/day,25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day, wherein the patientexhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10,IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof,e.g., IL-15, IP-10, and/or IL-7, or decreased serum levels of perforinand/or MIP-1b after the administration of the cyclophosphamide andfludarabine. In one embodiment, the invention includes a method ofconditioning a patient in need of a T cell therapy comprisingadministering to the patient a dose of cyclophosphamide equal to orhigher than about 30 mg/kg/day and lower than 60 mg/kg/day and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day, e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day.

In another embodiment, the invention includes a method of reducing ordepleting endogenous lymphocytes in a patient in need of a T celltherapy comprising administering to the patient a dose ofcyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/dayand a dose of fludarabine between about 20 m g/m²/day and about 900mg/m²/day.

In another embodiment, the invention includes a method of reducing ordepleting endogenous lymphocytes in a patient in need of a T celltherapy comprising administering to the patient a dose ofcyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/day(e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day) and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day), wherein thepatient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5,IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combinationthereof, e.g., IL-15, IP-10, and/or IL-7, or decreased serum levels ofperforin and/or MIP-1b after the administration of the cyclophosphamideand fludarabine. In one embodiment, the invention includes a method ofreducing or depleting endogenous lymphocytes in a patient in need of a Tcell therapy comprising administering to the patient a dose ofcyclophosphamide between about 1110 mg/m²/day and about 2000 mg/m²/dayand a dose of fludarabine between about 20 mg/m²/day and about 900mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60mg/m²/day), wherein the patient exhibits increased serum levels of IL-7,IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, orany combination thereof, e.g., IL-15, IP-10, and/or IL-7, or decreasedserum levels of perforin and/or MIP-1b after the administration of thecyclophosphamide and fludarabine. In one embodiment, the inventionincludes a method of reducing or depleting endogenous lymphocytes in apatient in need of a T cell therapy comprising administering to thepatient a dose of cyclophosphamide equal to or higher than 30 mg/kg/dayand lower than 60 mg/kg/day and a dose of fludarabine between about 20mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30mg/m²/day, or 60 mg/m²/day), wherein the patient exhibits increasedserum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP,sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and/orIL-7, or decreased serum levels of perforin and/or MIP-1b after theadministration of the cyclophosphamide and fludarabine.

In other embodiments, the invention includes a method of increasing theavailability of a homeostatic cytokine in a patient in need of a T celltherapy comprising administering to the patient a dose ofcyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/day(e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day) and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In anotherembodiment, the invention includes a method of increasing theavailability of a homeostatic cytokine in a patient in need of a T celltherapy comprising administering to the patient a dose ofcyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/day(e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day) and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day), wherein thepatient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5,IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combinationthereof, e.g., IL-15, IP-10, and/or IL-7, or decreased serum levels ofperforin and/or MIP-1b after the administration of the cyclophosphamideand fludarabine. In one embodiment, the invention includes a method ofincreasing the availability of a homeostatic cytokine in a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide between about 1110 mg/m²/day and about 2000mg/m²/day and a dose of fludarabine between about 20 mg/m²/day and about900 mg/m²/day (e.g., 20 m g/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60mg/m²/day), wherein the patient exhibits increased serum levels of IL-7,IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, orany combination thereof, e.g., IL-15, IP-10, and/or IL-7, or decreasedserum levels of perforin and/or MIP-1b after the administration of thecyclophosphamide and fludarabine. In one embodiment, the inventionincludes a method of increasing the availability of a homeostaticcytokine in a patient in need of a T cell therapy comprisingadministering to the patient a dose of cyclophosphamide equal to orhigher than about 30 mg/kg/day and lower than 60 mg/kg/day and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day), wherein thepatient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5,IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combinationthereof, e.g., IL-15, IP-10, and/or IL-7, or decreased serum levels ofperforin and/or MIP-1b after the administration of the cyclophosphamideand fludarabine.

In one particular embodiment, the invention includes a method ofenhancing an effector function of administered T cells in a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/day(e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day) and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In anotherembodiment, the invention includes a method of enhancing an effectorfunction of administered T cells in a patient in need of a T celltherapy comprising administering to the patient a dose ofcyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/day(e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day) and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day), wherein thepatient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5,IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combinationthereof, e.g., IL-15, IP-10, and/or IL-7, or decreased serum levels ofperforin and/or MIP-1b after the administration of the cyclophosphamideand fludarabine. In one embodiment, the invention includes a method ofenhancing an effector function of administered T cells in a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide between about 1110 mg/m²/day and about 2000mg/m²/day and a dose of fludarabine between about 20 mg/m²/day and about900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60mg/m²/day), wherein the patient exhibits increased serum levels of IL-7,IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, orany combination thereof, e.g., IL-15, IP-10, and/or IL-7, or decreasedserum levels of perforin and/or MIP-1b after the administration of thecyclophosphamide and fludarabine. In one embodiment, the inventionincludes a method of enhancing an effector function of administered Tcells in a patient in need of a T cell therapy comprising administeringto the patient a dose of cyclophosphamide equal to or higher than about30 mg/kg/day and lower than 60 mg/kg/day and a dose of fludarabinebetween about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day,25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day), wherein the patientexhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10,IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof,e.g., IL-15, IP-10, and/or IL-7, or decreased serum levels of perforinand/or MIP-1b after the administration of the cyclophosphamide andfludarabine.

In some embodiments, the invention includes a method of enhancingantigen presenting cell activation and/or availability in a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/day(e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day) and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In anotherembodiment, the invention includes a method of enhancing antigenpresenting cell activation and/or availability in a patient in need of aT cell therapy comprising administering to the patient a dose ofcyclophosphamide between about 200 mg/m²/day and about 2000 mg/m²/day(e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day) and a dose offludarabine between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day), wherein thepatient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5,IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combinationthereof, e.g., IL-15, IP-10, and/or IL-7, or decreased serum levels ofperforin and/or MIP-1b after the administration of the cyclophosphamideand fludarabine. In one embodiment, the invention includes a method ofenhancing antigen presenting cell activation and/or availability in apatient in need of a T cell therapy comprising administering to thepatient a dose of cyclophosphamide between about 1110 mg/m²/day andabout 2000 mg/m²/day and a dose of fludarabine between about 20mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30mg/m²/day, or 60 mg/m²/day), wherein the patient exhibits increasedserum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP,sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and/orIL-7, or decreased serum levels of perforin and/or MIP-1b after theadministration of the cyclophosphamide and fludarabine. In oneembodiment, the invention includes a method of enhancing antigenpresenting cell activation and/or availability in a patient in need of aT cell therapy comprising administering to the patient a dose ofcyclophosphamide equal to or higher than about 30 mg/kg/day and lowerthan 60 mg/kg/day and a dose of fludarabine between about 20 mg/m²/dayand about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day,or 60 mg/m²/day), wherein the patient exhibits increased serum levels ofIL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1,sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and/or IL-7, ordecreased serum levels of perforin and/or MIP-1b after theadministration of the cyclophosphamide and fludarabine.

The methods of the present invention include the administration ofcyclophosphamide and fludarabine prior to a T cell therapy. The timingof the administration of each component can be adjusted to maximizeeffect. As described herein, the day that a T cell therapy isadministered is designated as day 0. The cyclophosphamide andfludarabine can be administered at any time prior to administration ofthe T cell therapy. In some embodiments, the administration of thecyclophosphamide and fludarabine begins at least seven days, at leastsix days, at least five days, at least four days, at least three days,at least two days, or at least one day prior to the administration ofthe T cell therapy. In other embodiments, the administration of thecyclophosphamide and fludarabine begins at least eight days, at leastnine days, at least ten days, at least eleven days, at least twelvedays, at least thirteen days, or at least fourteen days prior to theadministration of the T cell therapy. In one embodiment, theadministration of the cyclophosphamide and fludarabine begins seven daysprior to the administration of the T cell therapy. In anotherembodiment, the administration of the cyclophosphamide and fludarabinebegins five days prior to the administration of the T cell therapy.

In one particular embodiment, administration of the cyclophosphamidebegins about seven days prior to the administration of the T celltherapy, and the administration of the fludarabine begins about fivedays prior to the administration of the T cell therapy. In anotherembodiment, administration of the cyclophosphamide begins about fivedays prior to the administration of the T cell therapy, and theadministration of the fludarabine begins about five days prior to theadministration of the T cell therapy.

The timing of the administration of each component can be adjusted tomaximize effect. In general, the cyclophosphamide and fludarabine can beadministered daily. In some embodiments, the cyclophosphamide andfludarabine are administered daily for about two days, for about threedays, for about four days, for about five days, for about six days, orfor about seven days. In one particular embodiment, the cyclophosphamideis administered daily for 2 days, and the fludarabine is administereddaily for five days. In another embodiment, both the cyclophosphamideand the fludarabine are administered daily for about 3 days.

As described herein, the day the T cell therapy is administered to thepatient is designated as day 0. In some embodiments, thecyclophosphamide is administered to the patient on day 7 and day 6 priorto day 0 (i.e., day −7 and day −6). In other embodiments, thecyclophosphamide is administered to the patient on day −5, day −4, andday −3. In some embodiments, the fludarabine is administered to thepatient on day −5, day −4, day −3, day −2, and day −1. In otherembodiments, the fludarabine is administered to the patient on day −5,day −4, and day −3.

The cyclophosphamide and fludarabine can be administered on the same ordifferent days. If the cyclophosphamide and fludarabine are administeredon the same day, the cyclophosphamide can be administered either beforeor after the fludarabine. In one embodiment, the cyclophosphamide isadministered to the patient on day −7 and day −6, and the fludarabine isadministered to the patient on day −5, day −4, day −3, day −2, and day−1. In another embodiment, the cyclophosphamide is administered to thepatient on day −5, day −4, and day −3, and the fludarabine isadministered to the patient on day −5, day −4, and day −3.

In certain embodiments, cyclophosphamide and fludarabine can beadministered concurrently or sequentially. In one embodiment,cyclophosphamide is administered to the patient prior to fludarabine. Inanother embodiment, cyclophosphamide is administered to the patientafter fludarabine.

The cyclophosphamide and fludarabine can be administered by any route,including intravenously (IV). In some embodiments, the cyclophosphamideis administered by IV over about 30 minutes, over about 35 minutes, overabout 40 minutes, over about 45 minutes, over about 50 minutes, overabout 55 minutes, over about 60 minutes, over about 90 minutes, overabout 120 minutes. In some embodiments, the fludarabine is administeredby IV over about 10 minutes, over about 15 minutes, over about 20minutes, over about 25 minutes, over about 30 minutes, over about 35minutes, over about 40 minutes, over about 45 minutes, over about 50minutes, over about 55 minutes, over about 60 minutes, over about 90minutes, over about 120 minutes.

In certain embodiments, a T cell therapy is administered to the patientfollowing administration of cyclophosphamide and fludarabine. In someembodiments, the T cell therapy comprises an adoptive cell therapy. Incertain embodiments, the adoptive cell therapy is selected fromtumor-infiltrating lymphocyte (TIL) immunotherapy, autologous celltherapy, engineered autologous cell therapy (eACT), and allogeneic Tcell transplantation. In one particular embodiment, the eACT comprisesadministration of engineered antigen specific chimeric antigen receptor(CAR) positive (+) T cells. In another embodiment, the eACT comprisesadministration of engineered antigen specific T cell receptor (TCR)positive (+) T cells. In some embodiments the engineered T cells treat atumor in the patient.

In one particular embodiment, the invention includes a method ofconditioning a patient in need of a T cell therapy comprisingadministering to the patient a dose of cyclophosphamide of about 500 mg/m²/day and a dose of fludarabine of about 60 mg/m²/day, wherein thecyclophosphamide is administered on days −5, −4, and −3, and wherein thefludarabine is administered on days −5, −4, and −3. In anotherembodiment, the invention includes a method of conditioning a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide of about 500 mg/m²/day and a dose of fludarabine ofabout 60 mg/m²/day, wherein the cyclophosphamide is administered on days−7 and −6, and wherein the fludarabine is administered on days −5, −4,−3, −2, and −1. In another embodiment, the invention includes a methodof conditioning a patient in need of a T cell therapy comprisingadministering to the patient a dose of cyclophosphamide of about 500mg/m²/day and a dose of fludarabine of about 30 mg/m²/day, wherein thecyclophosphamide is administered on days −7 and −6, and wherein thefludarabine is administered on days −5, −4, −3, −2, and −1. In anotherembodiment, the invention includes a method of conditioning a patient inneed of a T cell therapy comprising administering to the patient a doseof cyclophosphamide of about 300 mg/m²/day and a dose of fludarabine ofabout 60 mg/m²/day, wherein the cyclophosphamide is administered on days−7 and −6, and wherein the fludarabine is administered on days −5, −4,−3, −2, and −1.

Various other interventions may be included in the methods describedherein. For example, it is well known that cyclophosphamide andfludarabine may cause adverse events in patients followingadministration. It is within the scope of the invention thatcompositions may also be administered to the patient to reduce some ofthese adverse events. In some embodiments, the method further comprisesadministering a saline solution to the patient. The saline solution canbe administered to the patient either prior to or after theadministration of the cyclophosphamide and/or fludarabine, or bothbefore and after the administration of the cyclophosphamide and/orfludarabine. In certain embodiments, the saline solution can beadministered concurrently with the cyclophosphamide and/or fludarabine.In one particular embodiment, saline solution is administered to thepatient prior to the administration of cyclophosphamide and/orfludarabine and following the administration of cyclophosphamide and/orfludarabine on the day of each infusion.

The saline solution may be administered to the patient by any route,including, e.g., intravenously or orally. In some embodiments, themethod comprises administering about 0.1 L, about 0.2 L, about 0.3 L,about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about0.9 L, about 1 L, about 1.1 L, about 1.2 L, about 1.3 L, about 1.4 L,about 1.5 L, about 1.6 L, about 1.7 L, about 1.8 L, about 1.9 L, orabout 2.0 L of saline solution. The NaCl of the saline solution can bedissolved to a final concentration of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%. Inone embodiment, the method comprises administering 1.0 L of 0.9% NaClsaline solution to the patient. In one particular embodiment, the methodcomprises administering 1.0 L of 0.9% NaCl saline solution to thepatient prior to the administration of cyclophosphamide and/orfludarabine and following the administration of cyclophosphamide and/orfludarabine on the day of each infusion.

Further, adjuvants and excipients can also be administered to thepatient. For example, mesna (sodium 2-sulfanylthanesulfonate) is anadjuvant that acts as a detoxifying agent to inhibit hemorrhagiccystitis and hematuria, which can occur following treatment withcyclophosphamide. Cyclophosphamide, in vivo, can be converted tourotoxic metabolites, such as acrolein. Mesna assists to detoxify thesemetabolites by reaction of its sulfhydryl group with the vinyl group. Italso increases urinary excretion of cysteine. In certain embodiments,the method further comprises administering mesna to the patient. Themesna can be administered prior to the administration of thecyclophosphamide and/or fludarabine, after the administration of thecyclophosphamide and/or fludarabine, or both prior to and after theadministration of the of the cyclophosphamide and/or fludarabine. In oneembodiment, Mesna is administered intravenously or orally (per mouth).For example, oral mesna can be given with oral cyclophosphamide.

In addition, exogenous cytokines may also be administered to the patientin the method described herein. As discussed above, it is hypothesizedthat reducing the number of endogenous lymphocytes increases thebioavailability of endogenous molecules, such as cytokines, that canfavor the expansion, activation, and trafficking of adoptivelytransferred T cells. Accordingly, various cytokines may be administeredto the patient. In one embodiment, the method further comprisesadministering one or more doses of IL-2, IL-15, IL-7, IL-10, IL-5,IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combinationthereof. In one particular embodiment, the method comprisesadministering one or more doses of IL-2. The dose of IL-2 can be atleast about 10,000 IU/kg, at least about 50,000 IU/kg, at least about100,000 IU/kg, at least about 200,000 IU/kg, at least about 400,000IU/kg, at least about 600,000 IU/kg, at least about 700,000 IU/kg, atleast about 800,000 IU/kg, or at least about 1,000,000 IU/kg.

Cyclophosphamide and Fludarabine

Cyclophosphamide (ENDOXAN®, CYTOXAN®, PROCYTOX®, NEOSAR®, REVIMMUNE®,CYCLOBLASTIN®) is a nitrogen mustard-derivative alkylating agent withpotent immunosuppressive activity. Cyclophosphamide acts as anantineoplastic, and it is used to treat various types of cancersincluding lymphoma, multiple myeloma, leukemia, mycosis fungoides,neuroblastoma, ovarian cancer, eye cancer, and breast cancer, as well asautoimmune disorders.

Once administered to a patient, cyclophosphamide is converted intoacrolein and phosphoramide in the liver. Together, these metabolitescrosslink DNA in both resting and dividing cells by adding an alkylgroup to guanine bases of DNA at the number seven nitrogen atom of theimidazole ring. As a result, DNA replication is inhibited leading tocell death.

In the present invention, the dose of cyclophosphamide can be adjusteddepending on the desired effect, e.g., to modulate the reduction ofendogenous lymphocytes and/or control the severity of adverse events.For example, the dose of cyclophosphamide can be higher than about 300mg/m²/day and lower than about 900 mg/m²/day. In some embodiments, thedose of cyclophosphamide is about 350 mg/m²/day—about 2000 mg/m²/day, atleast about 400 mg/m²/day—about 2000 mg/m²/day, about 450mg/m²/day—about 2000 mg/m²/day, about 500 mg/m²/day—about 2000mg/m²/day, about 550 mg/m²/day—about 2000 mg/m²/day, or about 600mg/m²/day—about 2000 mg/m²/day. In another embodiment, the dose ofcyclophosphamide is about 350 mg/m²/day—about 1500 mg/m²/day, about 350mg/m²/day—about 1000 mg/m²/day, about 400 mg/m²/day—about 900 mg/m²/day,about 450 mg/m²/day—about 800 mg/m²/day, about 450 mg/m²/day—about 700mg/m²/day, about 500 mg/m²/day—about 600 mg/m²/day, or about 300mg/m²/day—about 500 mg/m²/day. In certain embodiments, the dose ofcyclophosphamide is about 350 mg/m²/day, about 400 mg/m²/day, about 450mg/m²/day, about 500 mg/m²/day, about 550 mg/m²/day, about 600mg/m²/day, about 650 mg/m²/day, about 700 mg/m²/day, about 800mg/m²/day, about 900 mg/m²/day, or about 1000 mg/m²/day. In oneparticular embodiment, the dose of cyclophosphamide is about 200mg/m²/day. In one particular embodiment, the dose of cyclophosphamide isabout 300 mg/m²/day. In another embodiment, the dose of cyclophosphamideis about 500 mg/m²/day. In other embodiments, the dose ofcyclophosphamide is about 200 mg/m²/day—about 2000 mg/m²/day, about 300mg/m²/day—about 2000 mg/m²/day, about 400 mg/m²/day—about 2000mg/m²/day, about 500 mg/m²/day—about 2000 mg/m²/day, about 600mg/m²/day—about 2000 mg/m²/day, about 700 mg/m²/day—about 2000mg/m²/day, about 800 mg/m²/day—about 2000 mg/m²/day, about 900mg/m²/day—about 2000 mg/m²/day, about 1000 mg/m²/day—about 2000mg/m²/day, about 1100 mg/m²/day—about 2000 mg/m²/day, about 1200mg/m²/day—about 2000 mg/m²/day, about 1300 mg/m²/day—about 2000mg/m²/day, about 1400 mg/m²/day—about 2000 mg/m²/day, about 1500mg/m²/day—about 2000 mg/m²/day, about 1600 mg/m²/day—about 2000mg/m²/day, about 1700 mg/m²/day—about 2000 mg/m²/day, about 1800mg/m²/day—about 2000 mg/m²/day, about 1900 mg/m²/day—about 2000mg/m²/day, about 200 mg/m²/day—about 1900 mg/m²/day, about 400mg/m²/day—about 1800 mg/m²/day, about 500 mg/m²/day—about 1700mg/m²/day, about 600 mg/m²/day—about 1600 mg/m²/day, about 700mg/m²/day—about 1500 mg/m²/day, about 800 mg/m²/day—about 1400mg/m²/day, about 900 mg/m²/day—about 1300 mg/m²/day, about 1000mg/m²/day—about 1200 mg/m²/day, about 1100 mg/m²/day—about 1200mg/m²/day, or about 1110 mg/m²/day—about 1150 mg/m²/day.

Fludarabine phosphate (FLUDARA®) is a synthetic purine nucleoside thatdiffers from physiologic nucleosides in that the sugar moiety isarabinose instead of ribose or deoxyribose. Fludarabine acts as a purineantagonist antimetabolite, and it is used to treat various types ofhematological malignancies, including various lymphomas and leukemias.

Once administered to a patient, fludarabine is rapidly dephosphorylatedto 2-fluoro-ara-A and then phosphorylated intracellularly bydeoxycytidine kinase to the active triphosphate, 2-fluoro-ara-ATP. Thismetabolite then interferes with DNA replication, likely by inhibitingDNA polymerase alpha, ribonucleotide reductase, and DNA primase, thusinhibiting DNA synthesis. As a result, fludarabine administration leadsto increased cell death in dividing cells.

In the present invention, the dose of fludarabine can also be adjusteddepending on the desired effect. For example, the dose of fludarabinecan be higher than 30 mg/m²/day and lower than 900 mg/m²/day. In someembodiments, the dose of fludarabine can be about 35 mg/m²/day—about 900mg/m²/day, about 40 mg/m²/day—about 900 mg/m²/day, about 45mg/m²/day—about 900 mg/m²/day, about 50 mg/m²/day—about 900 mg/m²/day,about 55 mg/m²/day—about 900 mg/m²/day, or about 60 mg/m²/day—about 900mg/m²/day. In other embodiments, the dose of fludarabine is about 35mg/m²/day—about 900 mg/m²/day, about 35 mg/m²/day—about 800 mg/m²/day,about 35 mg/m²/day—about 700 mg/m²/day, about 35 mg/m²/day—about 600mg/m²/day, about 35 mg/m²/day—about 500 mg/m²/day, about 35mg/m²/day—about 400 mg/m²/day, about 35 mg/m²/day—about 300 mg/m²/day,about 35 mg/m²/day—about 200 mg/m²/day, about 35 mg/m²/day—about 100mg/m²/day, about 40 mg/m²/day—about 90 mg/m²/day, about 45mg/m²/day—about 80 mg/m²/day, about 45 mg/m²/day—about 70 mg/m²/day, orabout 50 mg/m²/day—about 60 mg/m²/day. In certain embodiments, the doseof fludarabine is about 35 mg/m²/day, about 40 mg/m²/day, about 45mg/m²/day, about 50 mg/m²/day, about 55 mg/m²/day, about 60 mg/m²/day,about 65 mg/m²/day, about 70 mg/m²/day, about 75 mg/m²/day, about 80mg/m²/day, about 85 mg/m²/day, about 90 mg/m²/day, about 95 mg/m²/day,about 100 mg/m²/day, about 200 mg/m²/day, or about 300 mg/m²/day. Inother embodiments, the dose of fludarabine is about 110 mg/m²/day, 120mg/m²/day, 130 mg/m²/day, 140 mg/m²/day, 150 mg/m²/day, 160 mg/m²/day,170 mg/m²/day, 180 mg/m²/day, or 190 mg/m²/day. In some embodiments, thedose of fludarabine is about 210 mg/m²/day, 220 mg/m²/day, 230mg/m²/day, 240 mg/m²/day, 250 m g/m²/day, 260 mg/m²/day, 270 mg/m²/day,280 mg/m²/day, or 290 mg/m²/day. In one particular embodiment, the doseof fludarabine is about 20 mg/m²/day. In one particular embodiment, thedose of fludarabine is about 30 mg/m²/day. In another embodiment, thedose of fludarabine is about 60 mg/m²/day. In another embodiment, thedose of fludarabine is about 25 mg/m²/day.

The doses of cyclophosphamide and fludarabine can be raised or loweredtogether or independently. For example, the dose of cyclophosphamide canbe increased while the dose of fludarabine is decreased, and the dose ofcyclophosphamide can be decreased while the dose of fludarabine isincreased. Alternatively, the dose of both cyclophosphamide andfludarabine can be increased or decreased together.

In some embodiments, the dose of cyclophosphamide is 100 mg/m²/day (or110 mg/m²/day, 120 mg/m²/day, 130 mg/m²/day, or 140 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 150 mg/m²/day (or160 mg/m²/day, 170 mg/m²/day, 180 mg/m²/day, or 190 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is about 200 mg/m²/day(or 210 mg/m²/day, 220 mg/m²/day, 230 mg/m²/day, or 240 mg/m²/day) andthe dose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 250 mg/m²/day (or260 mg/m²/day, 270 mg/m²/day, 280 mg/m²/day, or 290 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 300 mg/m²/day (or310 mg/m²/day, 320 mg/m²/day, 330 mg/m²/day, or 340 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 350 mg/m²/day (or360 mg/m²/day, 370 mg/m²/day, 380 mg/m²/day, or 390 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 400 mg/m²/day (or410 mg/m²/day, 420 mg/m²/day, 430 mg/m²/day, or 440 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 450 mg/m²/day (or460 mg/m²/day, 470 mg/m²/day, 480 mg/m²/day, or 490 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 500 mg/m²/day (or510 mg/m²/day, 520 mg/m²/day, 530 mg/m²/day, or 540 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 550 mg/m²/day (or560 mg/m²/day, 570 mg/m²/day, 580 mg/m²/day, or 590 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 600 mg/m²/day (or610 mg/m²/day, 620 mg/m²/day, 630 mg/m²/day, or 640 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 650 mg/m²/day (or660 mg/m²/day, 670 mg/m²/day, 680 mg/m²/day, or 690 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 700 mg/m²/day (or710 mg/m²/day, 720 mg/m²/day, 730 mg/m²/day, or 740 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 750 mg/m²/day (or760 mg/m²/day, 770 mg/m²/day, 780 mg/m²/day, or 790 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 800 mg/m²/day (or810 mg/m²/day, 820 mg/m²/day, 830 mg/m²/day, or 840 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 850 mg/m²/day (or860 mg/m²/day, 870 mg/m²/day, 880 mg/m²/day, or 890 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 900 mg/m²/day (or910 mg/m²/day, 920 mg/m²/day, 930 mg/m²/day, or 940 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 950 mg/m²/day (or960 mg/m²/day, 970 mg/m²/day, 980 mg/m²/day, or 990 mg/m²/day) and thedose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 1000 mg/m²/day (or1010 mg/m²/day, 1020 mg/m²/day, 1030 mg/m²/day, or 1040 mg/m²/day) andthe dose of fludarabine is 5 mg/m²/day, 10 mg/m²/day, 15 mg/m²/day, 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, 35 mg/m²/day, 40 mg/m²/day, 45mg/m²/day, 50 mg/m²/day, 55 mg/m²/day, 60 mg/m²/day, 65 mg/m²/day, 70mg/m²/day, or 75 mg/m²/day.

In other embodiments, the dose of cyclophosphamide is between 100mg/m²/day and 650 mg/m²/day, and the dose of fludarabine is between 10mg/m²/day and 50 mg/m²/day. In other embodiments, the dose ofcyclophosphamide is between 150 mg/m²/day and 600 mg/m²/day, and thedose of fludarabine is between 20 mg/m²/day and 50 mg/m²/day. In otherembodiments, the dose of cyclophosphamide is between 200 mg/m²/day and550 mg/m²/day, and the dose of fludarabine is between 20 mg/m²/day and40 mg/m²/day. In other embodiments, the dose of cyclophosphamide isbetween 250 mg/m²/day and 550 mg/m²/day, and the dose of fludarabine isbetween 15 mg/m²/day and 45 mg/m²/day.

In certain embodiments, the dose of cyclophosphamide is 1000 mg/m²/day,and the dose of fludarabine is 60 mg/m²/day, 65 mg/m²/day, 70 mg/m²/day,75 mg/m²/day, 80 mg/m²/day, 85 mg/m²/day, 90 mg/m²/day, 95 mg/m²/day,100 mg/m²/day, 105 mg/m²/day, 110 mg/m²/day, 115 mg/m²/day, 120mg/m²/day, 125 mg/m²/day, 130 mg/m²/day, 135 mg/m²/day, 140 mg/m²/day,145 mg/m²/day, 150 mg/m²/day, 155 mg/m²/day, 160 mg/m²/day, 165mg/m²/day, 170 mg/m²/day, 175 mg/m²/day, 180 mg/m²/day, 185 mg/m²/day,190 mg/m²/day, 195 mg/m²/day, 200 mg/m²/day, 205 mg/m²/day, 210mg/m²/day, 215 mg/m²/day, 220 mg/m²/day, 225 mg/m²/day, 230 mg/m²/day,235 mg/m²/day, 240 mg/m²/day, 245 mg/m²/day, or 250 mg/m²/day.

In some embodiments, the dose of cyclophosphamide is 200 mg/m²/day andthe dose of fludarabine is 20 mg/m²/day. In some embodiments, the doseof cyclophosphamide is 200 mg/m²/day and the dose of fludarabine is 30mg/m²/day. In some embodiments, the dose of cyclophosphamide is 300mg/m²/day and the dose of fludarabine is 30 mg/m²/day. In otherembodiments, the dose of cyclophosphamide is 300 mg/m²/day and the doseof fludarabine is 60 mg/m²/day. In other embodiments, the dose ofcyclophosphamide is 500 mg/m²/day and the dose of fludarabine is 30mg/m²/day. In still other embodiments, the dose of cyclophosphamide is500 mg/m²/day and the dose of fludarabine is 60 mg/m²/day. In someembodiments, the dose of cyclophosphamide is about 1110 mg/m²/day andthe dose of fludarabine is 25 mg/m²/day. In some embodiments, the doseof cyclophosphamide is about 2000 mg/m²/day and the dose of fludarabineis 25 mg/m²/day. In some embodiments, the dose of cyclophosphamide is 30mg/kg/day and the dose of fludarabine is 25 mg/m²/day.

T Cell Therapy

The present invention provides methods of enhancing the effectiveness ofa T cell therapy by conditioning a patient by administering to thepatient cyclophosphamide and fludarabine. Because the conditioningregimens serve to modify the immune environment through induction ofmolecules that can favor the homeostatic expansion, activation, andtrafficking of T cells in general, various different T cell therapiescan benefit from the conditioning methods described herein. One of skillin the art would understand that the conditioning regimens could beapplied to any method of treating a patient comprising administering tothe patient one or more T cells.

For example, and without limitation, the conditioning regimens describedherein can enhance the effectiveness of a T cell therapy, which can bean adoptive T cell therapy selected from the group consisting oftumor-infiltrating lymphocyte (TIL) immunotherapy, autologous celltherapy, engineered autologous cell therapy (eACT), allogeneic T celltransplantation, non-T cell transplantation, and any combinationthereof. Adoptive T cell therapy broadly includes any method ofselecting, enriching in vitro, and administering to a patient autologousor allogeneic T cells that recognize and are capable of binding tumorcells. TIL immunotherapy is a type of adoptive T cell therapy, whereinlymphocytes capable of infiltrating tumor tissue are isolated, enrichedin vitro, and administered to a patient. The TIL cells can be eitherautologous or allogeneic. Autologous cell therapy is an adoptive T celltherapy that involves isolating T cells capable of targeting tumor cellsfrom a patient, enriching the T cells in vitro, and administering the Tcells back to the same patient. Allogeneic T cell transplantation caninclude transplant of naturally occurring T cells expanded ex vivo orgenetically engineered T cells. Engineered autologous cell therapy, asdescribed in more detail above, is an adoptive T cell therapy wherein apatient's own lymphocytes are isolated, genetically modified to expressa tumor targeting molecule, expanded in vitro, and administered back tothe patient. Non-T cell transplantation can include autologous orallogeneic therapies with non-T cells such as, but not limited to,natural killer (NK) cells.

In one particular embodiment, the T cell therapy of the presentinvention is engineered Autologous Cell Therapy (eACT™). According tothis embodiment, the method can include collecting blood cells from thepatient prior to the administration of cyclophosphamide and fludarabine.The isolated blood cells (e.g., T cells) can then be engineered toexpress a chimeric antigen receptor (“engineered CAR T cells”) or T cellreceptor (“engineered TCR T cells”). In a particular embodiment, theengineered CAR T cells or the engineered TCR T cells are administered tothe patient after administering the cyclophosphamide and fludarabine. Insome embodiments, the engineered T cells treat a tumor in the patient.

In one embodiment, the T cells can be engineered to express a chimericantigen receptor. The chimeric antigen receptor can comprise bindingmolecule to a tumor antigen. The binding molecule can be an antibody oran antigen binding molecule thereof. For example, the antigen bindingmolecule can be selected from scFv, Fab, Fab′, Fv, F(ab′)2, and dAb, andany fragments or combinations thereof.

The chimeric antigen receptor can further comprise a hinge region. Thehinge region can be derived from the hinge region of IgG1, IgG2, IgG3,IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha. In one particularembodiment, the hinge region is derived from the hinge region of IgG4.

The chimeric antigen receptor can also comprise a transmembrane domain.The transmembrane domain can be a transmembrane domain of anytransmembrane molecule that is a co-receptor on immune cells or atransmembrane domain of a member of the immunoglobulin superfamily. Incertain embodiments, the transmembrane domain is derived from atransmembrane domain of CD28, CD8 alpha, CD4, or CD19. In one particularembodiment, the transmembrane domain comprises a domain derived from aCD28 transmembrane domain.

The chimeric antigen receptor can further comprise one or morecostimulatory signaling regions. For example, the costimulatorysignaling region can be a signaling region of CD28, OX-40, 41BB, CD27,inducible T cell costimulator (ICOS), CD3 gamma, CD3 delta, CD3 epsilon,CD247, Ig alpha (CD79a), or Fc gamma receptor. In one particularembodiment, the costimulatory signaling region is a CD28 signalingregion.

In one embodiment, the chimeric antigen receptor further comprises a CD3zeta signaling domain.

The chimeric antigen receptor can be engineered to target a particulartumor antigen. In some embodiments, the tumor antigen is selected fromCD19 CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein,CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen,melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, folatebinding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelopeglycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met,mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4,ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 incombination, and any combination thereof. In one particular embodiment,the tumor antigen is CD19.

In another embodiment, the T cell therapy comprises administering to thepatient engineered T cells expressing T cell receptor (“engineered TCR Tcells”). The T cell receptor (TCR) can comprise a binding molecule to atumor antigen. In some embodiments, the tumor antigen is selected fromthe group consisting of CD19 CD20, ROR1, CD22, carcinoembryonic antigen,alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen,prostate-specific antigen, melanoma-associated antigen, mutated p53,mutated ras, HER2/Neu, folate binding protein, HIV-1 envelopeglycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33,CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha,kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 incombination, HER1-HER2 in combination, and any combination thereof.

In one embodiment, the TCR comprises a binding molecule to a viraloncogene. In one particular embodiment, the viral oncogene is selectedfrom human papilloma virus (HPV), Epstein-Barr virus (EBV), and humanT-lymphotropic virus (HTLV).

In still another embodiment, the TCR comprises a binding molecule to atesticular, placental, or fetal tumor antigen. In one particularembodiment, the testicular, placental, or fetal tumor antigen isselected from the group consisting of NY-ESO-1, synovial sarcoma Xbreakpoint 2 (SSX2), melanoma antigen (MAGE), and any combinationthereof.

In another embodiment, the TCR comprises a binding molecule to a lineagespecific antigen. In one particular embodiment, the lineage specificantigen is selected from the group consisting of melanoma antigenrecognized by T cells 1 (MART-1), gp100, prostate specific antigen(PSA), prostate specific membrane antigen (PSMA), prostate stem cellantigen (PSCA), and any combination thereof.

In one embodiment, the T cell therapy comprises administering to thepatient engineered CAR T cells expressing a chimeric antigen receptorthat binds to CD19 and further comprises a CD28 costimulatory domain anda CD3-zeta signaling region. In a particular embodiment, the T celltherapy comprises administering to a patient KTE-C19.

The T cell therapy included in the present invention involves thetransfer of T cells to a patient. The T cells can be administered at atherapeutically effective amount. For example, a therapeuticallyeffective amount of T cells, e.g., engineered CAR+ T cells or engineeredTCR+ T cells, can be at least about 10⁴ cells, at least about 10⁵ cells,at least about 10⁶ cells, at least about 10⁷ cells, at least about 10⁸cells, at least about 10⁹, or at least about 10¹⁰. In anotherembodiment, the therapeutically effective amount of the T cells, e.g.,engineered CAR+ T cells or engineered TCR+ T cells, is about 10⁴ cells,about 10⁵ cells, about 10⁶ cells, about 10⁷ cells, or about 10⁸ cells.In one particular embodiment, the therapeutically effective amount ofthe T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells,is about 1×10⁵ cells/kg, about 2×10⁵ cells/kg, about 3×10⁵ cells/kg,about 4×10⁵ cells/kg, about 5×10⁵ cells/kg, about 6×10⁵ cells/kg, about7×10⁵ cells/kg, about 8×10⁵ cells/kg, about 9×10⁵ cells/kg, about 1×10⁶cells/kg, about 2×10⁶ cells/kg, about 3×10⁶ cells/kg, about 4×10⁶cells/kg, about 5×10⁶ cells/kg, about 6×10⁶ cells/kg, about 7×10⁶cells/kg, about 8×10⁶ cells/kg, about 9×10⁶ cells/kg, about 1×10⁷cells/kg, about 2×10⁷ cells/kg, about 3×10⁷ cells/kg, about 4×10⁷cells/kg, about 5×10⁷ cells/kg, about 6×10⁷ cells/kg, about 7×10⁷cells/kg, about 8×10⁷ cells/kg, or about 9×10⁷ cells/kg. In oneparticular embodiment, the therapeutically effective amount of the Tcells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, isabout 2×10⁶ cells/kg.

In other embodiments, a therapeutically effective amount of T cells,e.g., engineered CAR+ T cells or engineered TCR+ T cells, is from about1.0×10⁵ cells/kg to about 2×10⁸ cells/kg, from about 2.0×10⁵ cells/kg toabout 2×10⁸ cells/kg, from about 3.0×10⁵ cells/kg to about 2×10⁸cells/kg, from about 4.0×10⁵ cells/kg to about 2×10⁸ cells/kg, fromabout 5.0×10⁵ cells/kg to about 2×10⁸ cells/kg, from about 6.0×10⁵cells/kg to about 2×10⁸ cells/kg, from about 7.0×10⁵ cells/kg to about2×10⁸ cells/kg, from about 8.0×10⁵ cells/kg to about 2×10⁸ cells/kg,from about 9.0×10⁵ cells/kg to about 2×10⁸ cells/kg, from about 0.5×10⁶cells/kg to about 2×10⁸ cells/kg, from about 2×10⁶ cells/kg to about9×10⁷ cells/kg, from about 3×10⁶ cells/kg to about 9×10⁷ cells/kg, fromabout 4×10⁶ cells/kg to about 9×10⁷ cells/kg, from about 5×10⁶ cells/kgto about 9×10⁷ cells/kg, from about 6×10⁶ cells/kg to about 9×10⁷cells/kg, from about 7×10⁶ cells/kg to about 9×10⁷ cells/kg, from about8×10⁶ cells/kg to about 9×10⁷ cells/kg, from about 9×10⁶ cells/kg toabout 9×10⁷ cells/kg, from about 1×10⁷ cells/kg to about 9×10⁷ cells/kg,from about 2×10⁷ cells/kg to about 9×10⁷ cells/kg, from about 3×10⁷cells/kg to about 9×10⁷ cells/kg, from about 4×10⁷ cells/kg to about9×10⁷ cells/kg, from about 5×10⁷ cells/kg to about 9×10⁷ cells/kg, fromabout 6×10⁷ cells/kg to about 9×10⁷ cells/kg, from about 7×10⁷ cells/kgto about 9×10⁷ cells/kg, from about 8×10⁷ cells/kg to about 9×10⁷cells/kg, from about 2×10⁶ cells/kg to about 8×10⁷ cells/kg, from about2×10⁶ cells/kg to about 7×10⁷ cells/kg, from about 2×10⁶ cells/kg toabout 6×10⁷ cells/kg, from about 2×10⁶ cells/kg to about 5×10⁷ cells/kg,from about 2×10⁶ cells/kg to about 4×10⁷ cells/kg, from about 2×10⁶cells/kg to about 3×10⁷ cells/kg, from about 2×10⁶ cells/kg to about2×10⁷ cells/kg, from about 2×10⁶ cells/kg to about 1×10⁷ cells/kg, fromabout 2×10⁶ cells/kg to about 9×10⁶ cells/kg, from about 2×10⁶ cells/kgto about 8×10⁶ cells/kg, from about 2×10⁶ cells/kg to about 7×10⁶cells/kg, from about 2×10⁶ cells/kg to about 6×10⁶ cells/kg, from about2×10⁶ cells/kg to about 5×10⁶ cells/kg, from about 2×10⁶ cells/kg toabout 4×10⁶ cells/kg, from about 2×10⁶ cells/kg to about 3×10⁶ cells/kg,from about 3×10⁶ cells/kg to about 8×10⁷ cells/kg, from about 4×10⁶cells/kg to about 7×10⁷ cells/kg, from about 5×10⁶ cells/kg to about6×10⁷ cells/kg, from about 6×10⁶ cells/kg to about 5×10⁷ cells/kg, fromabout 7×10⁶ cells/kg to about 4×10⁷ cells/kg, from about 8×10⁶ cells/kgto about 3×10⁷ cells/kg, or from about 9×10⁶ cells/kg to about 2×10⁷cells/kg. In one embodiment, the therapeutically effective amount of theengineered CAR T cells is from about 0.8×10⁶ cells/kg to about 1.2×10⁶ Tcells/kg. In one particular embodiment, the therapeutically effectiveamount of the engineered CAR T cells is 2.0×10⁵ cells/kg. In oneparticular embodiment, the therapeutically effective amount of theengineered CAR T cells is 1.0×10⁶ cells/kg.

Cytokine Levels

The invention describes a method of conditioning a patient in need of aT cell therapy comprising administering to the patient cyclophosphamideand fludarabine. Administration of cyclophosphamide and fludarabineprior to administration of a T cell therapy increases the level ofendogenous cytokines, modifying the immune environment in a way thatfavors the homeostatic expansion, activation and trafficking of T cells.Once the adoptively transferred T cells are administered to the patient,they are exposed to increased levels of endogenous cytokines.

Various cytokines can be enriched in patient serum followingcyclophosphamide and fludarabine administration. In some embodiments,the patient after the administration of cyclophosphamide and fludarabineand/or the T cell therapy exhibits an increased serum concentration of acytokine or a pro-inflammatory factor selected from interleukin (IL) 15,IL-7, IL-10, IL-5, IL-8, IL-1, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-9,IL-11, IL-12, IL-12p40, IL-12p70, IL-13, IL-14, IL-16, IL-17, IL-17a,IL-20, IL-21, granulocyte macrophage colony-stimulating factor (GM-CSF),granulocyte colony-stimulating factor (G-CSF), monocyte chemotacticprotein 1 (MCP-1), MCP-4, gamma-induced protein 10 (IP-10), placentalgrowth factor (PLGF), soluble intercellular adhesion molecule 1(sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), C-reactiveprotein (CRP), vascular endothelial growth factor (VEGF), VEGF-C,VEGF-D, macrophage inflammatory protein 1β (MIP-1β, MIP-1b), leukemiainhibitory factor (LIF), oncostatin M (OSM), interferon (IFN) alpha,IFN-beta, IFN-gamma, tumor necrosis factor (TNF) alpha, TNF-beta, CD154,lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), a proliferation-inducingligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-relatedligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14),OX40L, TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1),TNF-related apoptosis-inducing ligand (TRAIL), chemokine (C-C motif)ligand (CCL) 1, macrophage inflammatory protein 1 alpha (MIP-1a orCCL3), CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocytechemoattractant protein 2 (MCP-2 or CCL8), CCL13, thymus and activationregulated chemokine (TARC or CCL17), CCL22, FGF2, eotaxin, MDC, granzineA, granzine B, perforin, SAA, MCP-4, and any combination thereof. Insome embodiments, following the administration of cyclophosphamide andfludarabine the patient exhibits increased serum levels of IL-15 and/orIP-10. In some embodiments, following the administration ofcyclophosphamide and fludarabine the patient exhibits a decreased serumlevel of perforin.

In some embodiments, the invention includes a method of increasing theavailability of a homeostatic cytokine in a patient in need of a T celltherapy. In certain embodiments, the homeostatic cytokine is interleukin7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5(IL-5), gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), monocytechemotactic protein 1 (MCP-1), placental growth factor (PLGF),C-reactive protein (CRP), soluble intercellular adhesion molecule 1(sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or anycombination thereof.

In one embodiment, the serum level of IL-7 in the patient is increasedat least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, atleast 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, atleast 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, atleast 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, orat least 90 fold after the administration compared to the IL-7 serumlevel prior to the administration of cyclophosphamide and fludarabine.In a particular embodiment, the level of IL-7 is increased by at leastabout 2 fold compared to the IL-7 serum level prior to theadministration of cyclophosphamide and fludarabine. In anotherembodiment, the level of IL-7 is increased by administering exogenousIL-7 to the patient. In one particular embodiment, the level of IL-7 isincreased by administering to the patient cyclophosphamide, fludarabine,and exogenous IL-7.

In one embodiment, the serum level of IL-15 in the patient is increasedat least 5 fold, at least 10 fold, at least 15 fold, at least 20 fold,at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold,at least 45 fold, at least 50 fold, at least 60 fold, at least 70 fold,at least 80 fold, or at least 90 fold after the administration comparedto the IL-15 serum level prior to the administration of cyclophosphamideand fludarabine. In a particular embodiment, the level of IL-15 isincreased by at least about 10 fold compared to the IL-15 serum levelprior to the administration of cyclophosphamide and fludarabine. Inanother embodiment, the level of IL-15 is increased by at least about 20fold compared to the IL-15 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-15 is increased by at least about 30 fold compared to the IL-15 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IL-15 is increased by administeringexogenous IL-15 to the patient. In one particular embodiment, the levelof IL-15 is increased by administering to the patient cyclophosphamide,fludarabine, and exogenous IL-15.

In one embodiment, the serum level of IL-10 in the patient is increasedat least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, atleast 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, atleast 10 fold, or at least 20 fold after the administration compared tothe IL-10 serum level prior to the administration of cyclophosphamideand fludarabine. In a particular embodiment, the level of IL-10 isincreased by at least about 2 fold compared to the IL-10 serum levelprior to the administration of cyclophosphamide and fludarabine. Inanother embodiment, the level of IL-10 is increased by at least about 3fold compared to the IL-10 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-10 is increased by at least about 5 fold compared to the IL-10 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IL-10 is increased by at least about20 fold compared to the IL-10 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-10 is increased by administering exogenous IL-10 to the patient. Inone particular embodiment, the level of IL-10 is increased byadministering to the patient cyclophosphamide, fludarabine, andexogenous IL-10.

In one embodiment, the serum level of IL-5 in the patient is increasedat least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, atleast 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, atleast 10 fold, at least 15 fold, at least 20 fold, at least 30 fold, atleast 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, atleast 80 fold, at least 90 fold, or at least 100 fold after theadministration compared to the IL-5 serum level prior to theadministration of cyclophosphamide and fludarabine. In a particularembodiment, the level of IL-5 is increased by at least about 5 foldcompared to the IL-5 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-5 is increased by at least about 10 fold compared to the IL-5 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IL-5 is increased by at least about30 fold compared to the IL-5 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-5 is increased by at least about 100 fold compared to the IL-5 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IL-5 is increased by administeringexogenous IL-5 to the patient. In one particular embodiment, the levelof IL-5 is increased by administering to the patient cyclophosphamide,fludarabine, and exogenous IL-5.

In one embodiment, the serum level of IP-10 in the patient is increasedat least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, atleast 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, atleast 10 fold, at least 15 fold, at least 20 fold, or at least 30 foldafter the administration compared to the IP-10 serum level prior to theadministration of cyclophosphamide and fludarabine. In a particularembodiment, the level of IP-10 is increased by at least about 2 foldcompared to the IP-10 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIP-10 is increased by at least about 3 fold compared to the IP-10 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IP-10 is increased by at least about4 fold compared to the IP-10 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIP-10 is increased by at least about 7 fold compared to the IP-10 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IP-10 is increased by administeringexogenous IP-10 to the patient. In one particular embodiment, the levelof IP-10 is increased by administering to the patient cyclophosphamide,fludarabine, and exogenous IP-10.

In one embodiment, the serum level of IL-8 in the patient is increasedat least 2 fold, at least 5 fold, at least 10 fold, at least 15 fold, atleast 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, atleast 40 fold, at least 45 fold, at least 50 fold, at least 60 fold, atleast 70 fold, at least 80 fold, at least 90 fold, or at least 100 foldafter the administration compared to the IL-8 serum level prior to theadministration of cyclophosphamide and fludarabine. In a particularembodiment, the level of IL-8 is increased by at least about 2 foldcompared to the IL-8 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-8 is increased by at least about 5 fold compared to the IL-8 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IL-8 is increased by at least about10 fold compared to the IL-8 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-8 is increased by at least about 20 fold compared to the IL-8 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IL-8 is increased by at least about40 fold compared to the IL-8 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofIL-8 is increased by at least about 60 fold compared to the IL-8 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of IL-8 is increased by administeringexogenous IL-8 to the patient. In one particular embodiment, the levelof IL-8 is increased by administering to the patient cyclophosphamide,fludarabine, and exogenous IL-8.

In one embodiment, the serum level of MCP-1 in the patient is increasedat least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 15 fold, or at least 20 foldafter the administration compared to the MCP-1 serum level prior to theadministration of cyclophosphamide and fludarabine. In a particularembodiment, the level of MCP-1 is increased by at least about 2 foldcompared to the MCP-1 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofMCP-1 is increased by at least about 3 fold compared to the MCP-1 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of MCP-1 is increased by at least about5 fold compared to the MCP-1 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofMCP-1 is increased by at least about 7 fold compared to the MCP-1 serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of MCP-1 is increased by administeringexogenous MCP-1 to the patient. In one particular embodiment, the levelof MCP-1 is increased by administering to the patient cyclophosphamide,fludarabine, and exogenous MCP-1.

In one embodiment, the serum level of PLGF in the patient is increasedat least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 10 fold, at least 15 fold, at least 20 fold, atleast 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, atleast 45 fold, at least 50 fold, at least 60 fold, at least 70 fold, atleast 80 fold, at least 90 fold, or at least 100 fold after theadministration compared to the PLGF serum level prior to theadministration of cyclophosphamide and fludarabine. In a particularembodiment, the level of PLGF is increased by at least about 1.5 foldcompared to the PLGF serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofPLGF is increased by at least about 2 fold compared to the PLGF serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of PLGF is increased by at least about3 fold compared to the PLGF serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofPLGF is increased by administering exogenous PLGF to the patient. In oneparticular embodiment, the level of PLGF is increased by administeringto the patient cyclophosphamide, fludarabine, and exogenous PLGF.

In one embodiment, the serum level of CRP in the patient is increased atleast 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least about 9 fold, at least 10 fold, at least 15 fold,at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold,at least 40 fold, at least 45 fold, at least 50 fold, at least 60 fold,at least 70 fold, at least 80 fold, at least 90 fold, or at least 100fold after the administration compared to the CRP serum level prior tothe administration of cyclophosphamide and fludarabine. In a particularembodiment, the level of CRP is increased by at least about 1.5 foldcompared to the CRP serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofCRP is increased by at least about 2 fold compared to the CRP serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of CRP is increased by at least about 5fold compared to the CRP serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofCRP is increased by at least about 9 fold compared to the CRP serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of CRP is increased by at least about10 fold compared to the CRP serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofCRP is increased by at least about 25 fold compared to the CRP serumlevel prior to the administration of cyclophosphamide and fludarabine.In another embodiment, the level of CRP is increased by administeringexogenous CRP to the patient. In one particular embodiment, the level ofCRP is increased by administering to the patient cyclophosphamide,fludarabine, and exogenous CRP.

In one embodiment, the serum level of sICAM-1 in the patient isincreased at least 1.5 fold, at least 2 fold, at least 3 fold, at least4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20fold, at least 25 fold, or at least 30 fold after the administrationcompared to the sICAM-1 serum level prior to the administration ofcyclophosphamide and fludarabine. In a particular embodiment, the levelof sICAM-1 is increased by at least about 1.5 fold compared to thesICAM-1 serum level prior to the administration of cyclophosphamide andfludarabine. In another embodiment, the level of sICAM-1 is increased byat least about 2 fold compared to the sICAM-1 serum level prior to theadministration of cyclophosphamide and fludarabine. In anotherembodiment, the level of sICAM-1 is increased by at least about 3 foldcompared to the sICAM-1 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofsICAM-1 is increased by at least about 4 fold compared to the sICAM-1serum level prior to the administration of cyclophosphamide andfludarabine. In another embodiment, the level of sICAM-1 is increased byadministering exogenous sICAM-1 to the patient. In one particularembodiment, the level of sICAM-1 is increased by administering to thepatient cyclophosphamide, fludarabine, and exogenous sICAM-1.

In one embodiment, the serum level of sVCAM-1 in the patient isincreased at least 1.5 fold, at least 2 fold, at least 2.5 fold, atleast 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, orat least 5 fold after the administration compared to the sVCAM-1 serumlevel prior to the administration of cyclophosphamide and fludarabine.In a particular embodiment, the level of sVCAM-1 is increased by atleast about 1.5 fold compared to the sVCAM-1 serum level prior to theadministration of cyclophosphamide and fludarabine. In anotherembodiment, the level of sVCAM-1 is increased by at least about 2 foldcompared to the sVCAM-1 serum level prior to the administration ofcyclophosphamide and fludarabine. In another embodiment, the level ofsVCAM-1 is increased by at least about 3 fold compared to the sVCAM-1serum level prior to the administration of cyclophosphamide andfludarabine. In another embodiment, the level of sVCAM-1 is increased byadministering exogenous sVCAM-1 to the patient. In one particularembodiment, the level of sVCAM-1 is increased by administering to thepatient cyclophosphamide, fludarabine, and exogenous sVCAM-1.

In some embodiments, the level of one or more cytokine afteradministration of cyclophosphamide and fludarabine can be used to bepredict how a patient will respond to a T cell therapy. For example, anincrease in a particular cytokine following administration ofcyclophosphamide and fludarabine can indicate that a patient is morelikely to respond to a T cell therapy. In another example, a decrease orno change in the level of a particular cytokine following administrationwith cyclophosphamide and fludarabine can indicate that a patient isless likely to respond to a T cell therapy. It is also possible that anincrease in one or more cytokines and a decrease in one or moredifferent cytokines following administration of cyclophosphamide andfludarabine can indicate that a patient is more or less likely torespond to a T cell therapy. In that way, a patient's cytokine profilecan be indicative of responsiveness to a T cell therapy.

In some embodiments, a more than about 3 fold, a more than about 4 fold,a more than about 5 fold, a more than about 10 fold, a more than about15 fold, or a more than about 20 fold increase in IL-15 levels followingadministration of cyclophosphamide and fludarabine indicates that apatient will be more likely to respond to a T cell therapy. In otherembodiments, a more than about 2 fold, a more than about 3 fold, a morethan about 4 fold, a more than about 5 fold, or a more than about 6 foldincrease in IP-10 levels following administration of cyclophosphamideand fludarabine indicates that a patient will be more likely to respondto a T cell therapy. In still another embodiment, a decrease in MIP-1blevels following administration of cyclophosphamide and fludarabineindicates that a patient will be less likely to respond to a T celltherapy.

In some embodiments, the serum level of any one or more cytokine ismeasured one or more days before administration of cyclophosphamide andfludarabine and on one or more days selected from the day ofadministration of cyclophosphamide and fludarabine to 21 days afteradministration of cyclophosphamide and fludarabine.

One embodiment of the invention includes a method of increasing theavailability of a homeostatic cytokine in a patient in need of a T celltherapy. Another embodiment of the invention includes a method ofimproving the effect of a T cell therapy comprising administering to apatient a treatment that increases the level of one or more homeostatic,pro-inflammatory cytokine or chemokine selected from IL-15, IL-7, IL-10,IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, and sVCAM-1. One of skillin the art would recognize that the level of homeostatic cytokines canbe increased by a number of different methods, including but not limitedto, the use of cyclophosphamide and fludarabine as described herein,administration of one or more exogenous cytokines to the patient,administration of one or more composition that induces the expression ofor prevents the degradation of one or more endogenous cytokine,administration of one or more transgenic cells capable of expressing oneor more recombinant cytokines, and any other method having the effect ofincreasing the level of homeostatic cytokines in a patient.

In some embodiments, the invention includes a method of conditioning apatient in need of a T cell therapy, comprising administering to thepatient cyclophosphamide and fludarabine and one or more doses of anisolated or recombinant cytokine. The isolated or recombinant cytokinecan be any cytokine. In one embodiment, the cytokine is a homeostaticcytokine. In another embodiment, the cytokine is a pro-inflammatorycytokine. In still another embodiment, the cytokine is a chemokine. Inone particular embodiment, the method of conditioning a patient in needof a T cell therapy comprises administering to the patientcyclophosphamide and fludarabine and one or more doses of an isolated orrecombinant cytokine, wherein the cytokine is selected from IL-2, IL-15,IL-7, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, andany combination thereof, e.g., IL-15, IL-7, IP-10, MCP-1, CRP, and PLGF.The one or more doses of an isolated or recombinant cytokine can beadministered before the T cell therapy, or after the T Cell therapy, orany combination thereof.

In one embodiment, the method of conditioning a patient in need of a Tcell therapy, comprises administering to the patient cyclophosphamideand fludarabine and one or more doses IL-2. In some embodiments, thedose of IL-2 is at least about 10,000 IU/kg, at least about 50,000IU/kg, at least about 100,000 IU/kg, at least about 200,000 IU/kg, atleast about 400,000 IU/kg, at least about 600,000 IU/kg, at least about700,000 IU/kg, at least about 800,000 IU/kg, or at least about 1,000,000IU/kg. In one embodiment, the dose of IL-2 is at least about 700,000IU/kg. In one particular embodiment, the dose of IL-2 is about 720,000IU/kg. In some embodiments, IL-2 will be administered to the patientevery 8 hours until 15 doses or toxicity precludes additional doses.

Cancer Treatment

The methods of the invention can be used to treat a cancer in a subject,reduce the size of a tumor, kill tumor cells, prevent tumor cellproliferation, prevent growth of a tumor, eliminate a tumor from apatient, prevent relapse of a tumor, prevent tumor metastasis, induceremission in a patient, or any combination thereof. In certainembodiments, the methods induce a complete response. In otherembodiments, the methods induce a partial response.

Cancers that may be treated include tumors that are not vascularized,not yet substantially vascularized, or vascularized. The cancer may alsoinclude solid or non-solid tumors. In certain embodiments, the cancercan be selected from a tumor derived from bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, T-cell rich B cell lymphoma (TCRBCL),Primary mediastinal large B cell lymphoma (PMBCL), non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemia,acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers.

In one embodiment, the method can be used to treat a tumor, wherein thetumor is a lymphoma or a leukemia. Lymphoma and leukemia are cancers ofthe blood that specifically affect lymphocytes. All leukocytes in theblood originate from a single type of multipotent hematopoietic stemcell found in the bone marrow. This stem cell produces both myeloidprogenitor cells and lymphoid progenitor cell, which then give rise tothe various types of leukocytes found in the body. Leukocytes arisingfrom the myeloid progenitor cells include T lymphocytes (T cells), Blymphocytes (B cells), natural killer cells, and plasma cells.Leukocytes arising from the lymphoid progenitor cells includemegakaryocytes, mast cells, basophils, neutrophils, eosinophils,monocytes, and macrophages. Lymphomas and leukemias can affect one ormore of these cell types in a patient.

In general, lymphomas can be divided into at least two sub-groups:Hodgkin lymphoma and non-Hodgkin lymphoma. Non-Hodgkin Lymphoma (NHL) isa heterogeneous group of cancers originating in B lymphocytes, Tlymphocytes or natural killer cells. In the United States, B celllymphomas represent 80-85% of cases reported. In 2013 approximately69,740 new cases of NHL and over 19,000 deaths related to the diseasewere estimated to occur. Non-Hodgkin lymphoma is the most prevalenthematological malignancy and is the seventh leading site of new cancersamong men and women and account for 4% of all new cancer cases and 3% ofdeaths related to cancer.

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of NHL,accounting for approximately 30% of NHL cases. There are approximately22,000 new diagnoses of DLBCL in the United States each year. It isclassified as an aggressive lymphoma with the majority of patients curedwith conventional chemotherapy (NCCN guidelines NHL 2014).

First line therapy for DLBCL typically includes ananthracycline-containing regimen with rituximab, such as R-CHOP(rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone),which has an objective response rate of about 80% and a completeresponse rate of about 50% (Coiffier 2002), with about one-third ofpatients have refractory disease to initial therapy or relapse afterR-CHOP (Sehn 2005). For those patients who relapse after response tofirst line therapy, approximately 40-60% of patients can achieve asecond response with additional chemotherapy. The standard of care forsecond-line therapy for autologous stem cell transplant (ASCT) eligiblepatients includes rituximab and combination chemotherapy such as R-ICE(rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP(rituximab, dexamethasone, cytarabine, and cisplatin), which each havean objective response rate of about 63% and a complete response rate ofabout 26% (Gisselbrecht 2010). Patients who respond to second linetherapy and who are considered fit enough for transplant receiveconsolidation with high-dose chemotherapy and ASCT, which is curative inabout half of transplanted patients (Gisselbrecht 2010). Patients whofailed ASCT have a very poor prognosis and no curative options.

Primary mediastinal large B cell lymphoma (PMBCL) has distinct clinical,pathological, and molecular characteristics compared to DLBCL. PMBCL isthought to arise from thymic (medullary) B cells and representsapproximately 3% of patients diagnosed with DLBCL. PMBCL is typicallyidentified in the younger adult population in the fourth decade of lifewith a slight female predominance. Gene expression profiling suggestsderegulated pathways in PMBCL overlap with Hodgkin lymphoma. Initialtherapy of PMBCL generally includes anthracycline-containing regimenswith rituximab, such as infusional dose-adjusted etoposide, doxorubicin,and cyclophosphamide with vincristine, prednisone, and rituximab(DA-EPOCH-R), with or without involved field radiotherapy.

Follicular lymphoma (FL), a B cell lymphoma, is the most common indolent(slow-growing) form of NHL, accounting for approximately 20% to 30% ofall NHLs. Some patients with FL will transform (TFL) histologically toDLBCL which is more aggressive and associated with a poor outcome.Histological transformation to DLBCL occurs at an annual rate ofapproximately 3% for 15 years with the risk of transformation continuingto drop in subsequent years. The biologic mechanism of histologictransformation is unknown. Initial treatment of TFL is influenced byprior therapies for follicular lymphoma but generally includesanthracycline-containing regimens with rituximab to eliminate theaggressive component of the disease.

Treatment options for relapsed/refractory PMBCL and TFL are similar tothose in DLBCL. Given the low prevalence of these diseases, no largeprospective randomized studies in these patient populations have beenconducted. Patients with chemotherapy refractory disease have a similaror worse prognosis to those with refractory DLBCL.

In summary, subjects who have refractory, aggressive NHL (e.g., DLBCL,PMBCL and TFL) have a major unmet medical need and further research withnovel treatments are warranted in these populations.

Accordingly, in some embodiments, the method can be used to treat alymphoma or a leukemia, wherein the lymphoma or leukemia is a B cellmalignancy. In some embodiments, the lymphoma or leukemia is selectedfrom B-cell chronic lymphocytic leukemia/small cell lymphoma, B-cellprolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenströmmacroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia,plasma cell neoplasms (e.g., plasma cell myeloma (i.e., multiplemyeloma), or plasmacytoma), extranodal marginal zone B cell lymphoma(e.g., MALT lymphoma), nodal marginal zone B cell lymphoma, follicularlymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneousfollicle center lymphoma, mantle cell lymphoma, diffuse large B celllymphoma (DLBCL), Epstein-Barr virus-positive DLBCL, lymphomatoidgranulomatosis, primary mediastinal (thymic) large B-cell lymphoma(PMBCL), Intravascular large B-cell lymphoma, ALK+ large B-celllymphoma, plasmablastic lymphoma, primary effusion lymphoma, largeB-cell lymphoma arising in HHV8-associated multicentric Castleman'sdisease, Burkitt lymphoma/leukemia, T-cell prolymphocytic leukemia,T-cell large granular lymphocyte leukemia, aggressive NK cell leukemia,adult T-cell leukemia/lymphoma, extranodal NK/T-cell lymphoma,enteropathy-associated T-cell lymphoma, Hepatosplenic T-cell lymphoma,blastic NK cell lymphoma, Mycosis fungoides/Sezary syndrome, Primarycutaneous anaplastic large cell lymphoma, Lymphomatoid papulosis,Peripheral T-cell lymphoma, Angioimmunoblastic T cell lymphoma,Anaplastic large cell lymphoma, B-lymphoblastic leukemia/lymphoma,B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities,T-lymphoblastic leukemia/lymphoma, and Hodgkin lymphoma. In someembodiments, the cancer is refractory to one or more prior treatments,and/or the cancer has relapsed after one or more prior treatments.

In certain embodiments, the cancer is selected from follicular lymphoma,transformed follicular lymphoma, diffuse large B cell lymphoma, andprimary mediastinal (thymic) large B-cell lymphoma. In one particularembodiment, the cancer is diffuse large B cell lymphoma.

In some embodiments, the cancer is refractory to or the cancer hasrelapsed following one or more of chemotherapy, radiotherapy,immunotherapy (including a T cell therapy and/or treatment with anantibody or antibody-drug conjugate), an autologous stem celltransplant, or any combination thereof. In one particular embodiment,the cancer is refractory diffuse large B cell lymphoma.

In one particular embodiment, the invention includes a method oftreating a patient having a lymphoma comprising administering daily tothe patient cyclophosphamide at any dose described herein (e.g., about200 mg/m²/day, about 300 mg/m²/day, about 400 mg/m²/day, about 500mg/m²/day, about 600 mg/m²/day, about 700 mg/m²/day, about 800mg/m²/day, or about 900 mg/m²/day) and fludarabine at any dose describedherein (e.g., about 20 mg/m²/day, about 25 mg/m²/day, about 30mg/m²/day, about 35 mg/m²/day, about 40 mg/m²/day, about 45 mg/m²/day,about 50 mg/m²/day, about 55 mg/m²/day, about 60 mg/m²/day) for threedays prior to administration of a therapeutically effective amount ofengineered CAR cells to the patient, wherein the engineered CAR cellsexpress a chimeric antigen receptor that binds to CD19 and furthercomprises a CD28 costimulatory domain and a CD3-zeta signaling region.

In another embodiment, the invention includes a method of treating apatient having a lymphoma comprising (i) administering to the patientcyclophosphamide at any dose described herein (e.g., about 200mg/m²/day, about 300 mg/m²/day, about 400 mg/m²/day, about 500mg/m²/day, about 600 mg/m²/day, about 700 mg/m²/day, about 800mg/m²/day, or about 900 mg/m²/day) and fludarabine at any dose describedherein (e.g., about 20 mg/m²/day, about 25 mg/m²/day, about 30mg/m²/day, about 35 mg/m²/day, about 40 mg/m²/day, about 45 mg/m²/day,about 50 mg/m²/day, about 55 mg/m²/day, about 60 mg/m²/day) and (ii)administering to the patient a therapeutically effective amount ofengineered CAR cells, wherein the engineered CAR cells express achimeric antigen receptor that binds to CD19 and further comprises aCD28 costimulatory domain and a CD3-zeta signaling region.

In still another embodiment, the invention includes a method of treatinga patient having a lymphoma comprising administering to the patient atherapeutically effective amount of engineered CAR cells, wherein thepatient has been conditioned by administration of cyclophosphamide atany dose described herein (e.g., about 200 mg/m²/day, about 300mg/m²/day, about 400 mg/m²/day, about 500 mg/m²/day, about 600mg/m²/day, about 700 mg/m²/day, about 800 mg/m²/day, or about 900mg/m²/day) and fludarabine at any dose described herein (e.g., about 20mg/m²/day, about 25 mg/m²/day, about 30 mg/m²/day, about 35 mg/m²/day,about 40 mg/m²/day, about 45 mg/m²/day, about 50 mg/m²/day, about 55mg/m²/day, about 60 mg/m²/day) and wherein the engineered CAR cellsexpress a chimeric antigen receptor that binds to CD19 and furthercomprises a CD28 costimulatory domain and a CD3-zeta signaling region.

Kits

Also included within the scope of the present invention are kits, e.g.,pharmaceutical kits, comprising cyclophosphamide and fludarabine forpreconditioning uses for a T cell therapy. Kits typically include alabel indicating the intended use of the contents of the kit andinstructions for use. The term “label” includes any writing, or recordedmaterial supplied on or with the kit, or which otherwise accompanies thekit.

In some embodiments, the invention provides a kit conditioning a patientin need of a T cell therapy, the kit comprising: (i) cyclophosphamide,(ii) fludarabine, and (iii) instructions to administer cyclophosphamideat any dose described herein (e.g., between 200 mg/m²/day and 2000mg/m²/day) and fludarabine at any dose described herein (e.g., between20 mg/m²/day and 900 mg/m²/day) daily for three days to a patient inneed of an engineered CAR cell therapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at any dose described herein (e.g., between 200mg/m²/day and 2000 mg/m²/day) for two days from day −7 to day −6 andfludarabine at any dose described herein (e.g., between 20 mg/m²/day and900 mg/m²/day) daily for five days from day −5 to day −1 to a patient inneed of an engineered CAR cell therapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at any dose described herein (e.g., between 200mg/m²/day and 2000 mg/m²/day) and fludarabine at any dose describedherein (e.g., between 20 mg/m²/day and 900 mg/m²/day) daily for threedays to a patient in need of an engineered TCR cell therapy prior to thetherapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at any dose described herein (e.g., between 200mg/m²/day and 2000 mg/m²/day) for two days from day −7 to day −6 andfludarabine at any dose described herein (e.g., between 20 mg/m²/day and900 mg/m²/day) daily for five days from day −5 to day −1 to a patient inneed of an engineered TCR cell therapy prior to the therapy.

In some embodiments, the invention provides a kit conditioning a patientin need of a T cell therapy, the kit comprising: (i) cyclophosphamide,(ii) fludarabine, and (iii) instructions to administer cyclophosphamideat a dose of 300 mg/m²/day and fludarabine at a dose of 30 mg/m²/daydaily for three days to a patient in need of an engineered CAR celltherapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at a dose between 300 mg/m²/day for two days from day−7 to day −6 and fludarabine at a dose of 30 mg/m²/day daily for fivedays from day −5 to day −1 to a patient in need of an engineered CARcell therapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at a dose of 500 mg/m²/day and fludarabine at a dose of30 mg/m²/day daily for three days to a patient in need of an engineeredTCR cell therapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide a at a dose of 500 mg/m²/day for two days from day −7to day −6 and fludarabine at a dose of 30 mg/m²/day daily for five daysfrom day −5 to day −1 to a patient in need of an engineered TCR celltherapy prior to the therapy.

In some embodiments, the invention provides a kit conditioning a patientin need of a T cell therapy, the kit comprising: (i) cyclophosphamide,(ii) fludarabine, and (iii) instructions to administer cyclophosphamideat a dose of 300 mg/m²/day and fludarabine at a dose of 60 mg/m²/daydaily for three days to a patient in need of an engineered CAR celltherapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at a dose between 300 mg/m²/day for two days from day−7 to day −6 and fludarabine at a dose of 60 mg/m²/day daily for fivedays from day −5 to day −1 to a patient in need of an engineered CARcell therapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide at a dose of 500 mg/m²/day and fludarabine at a dose of60 mg/m²/day daily for three days to a patient in need of an engineeredTCR cell therapy prior to the therapy.

In other embodiments, the invention provides a kit conditioning apatient in need of a T cell therapy, the kit comprising: (i)cyclophosphamide, (ii) fludarabine, and (iii) instructions to administercyclophosphamide a at a dose of 500 mg/m²/day for two days from day −7to day −6 and fludarabine at a dose of 60 mg/m²/day daily for five daysfrom day −5 to day −1 to a patient in need of an engineered TCR celltherapy prior to the therapy.

In certain embodiments, the kit further comprises a saline solution andinstructions to administer the saline solution to the patient eitherprior to or after the administration of the cyclophosphamide and/orfludarabine, or both before and after the administration of thecyclophosphamide and/or fludarabine. In some embodiments, the kitfurther comprises mesna and instructions to administer the mesna to thepatient prior to the administration of the cyclophosphamide and/orfludarabine, after the administration of the cyclophosphamide and/orfludarabine, or both prior to and after the administration of the of thecyclophosphamide and/or fludarabine.

Diagnostics Using Biomarkers

The invention also includes methods of identifying a subject that issuitable for a T cell therapy. In one embodiment, the invention includesa method for treating a cancer in a patient suitable for a T celltherapy comprising preconditioning the patient by administering to thepatient cyclophosphamide at a dose between 200 mg/m² and 2000 mg/m²,e.g., 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m²,800 mg/m², 900 mg/m², 1000 mg/m², or 1110 mg/m², and fludarabine at adose between 20 mg/m² and 900 mg/m², e.g., 20 mg/m², 25 mg/m², 30 mg/m²,35 mg/m², 40 mg/m², 45 mg/m², or 50 mg/m², wherein the patient istreated with a T cell therapy after exhibiting an increased serum levelof IL-15, IP-10, and/or IL-7 and/or a decreased serum level of perforin.In another embodiment, the invention includes a method for treating acancer in a patient suitable for a T cell therapy comprising (i)preconditioning the patient by administering to the patientcyclophosphamide at a dose between 200 mg/m² and 2000 mg/m², e.g., 200mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m², 800 mg/m²,900 mg/m², 1000 mg/m², or 1110 mg/m², and fludarabine at a dose between20 mg/m² and 900 mg/m², e.g., 20 mg/m², 25 mg/m², 30 mg/m², 35 mg/m², 40mg/m², 45 mg/m², or 50 mg/m², and (ii) administering a T cell therapyafter the patient exhibits an increased serum level of IL-15, IP-10,and/or IL-7 and/or a decreased serum level of perforin. In otherembodiments, the invention is directed to a method for treating a cancerin a patient suitable for a T cell therapy comprising (i)preconditioning the patient by administering to the patientcyclophosphamide at a dose between 200 mg/m² and 2000 mg/m², e.g., 200mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m², 800 mg/m²,900 mg/m², 1000 mg/m², or 1110 mg/m², and fludarabine at a dose between20 mg/m² and 900 mg/m², e.g., 20 mg/m², 25 mg/m², 30 mg/m², 35 mg/m², 40mg/m², 45 mg/m², or 50 mg/m², (ii) administering an additional amount ofcyclophosphamide and/or fludarabine or administering IL-15, IP-10,and/or IL-7 when the patient does not exhibit sufficient serum levels ofIL-15, IP-10, and/or IL-7 after the administration in (i), and (iii)administering a T cell therapy after the patient exhibits an increasedserum level of IL-15, IP-10, and/or IL-7 after the administration in(ii). In certain embodiments, the T cell therapy is administered to thepatient when the patient exhibits an increased serum level of at leastone additional cytokine selected from the group consisting of MCP-1,CRP, PLGF, IP-10, and any combination thereof.

The invention further provides a method for identifying a patientsuitable for a T cell therapy comprising administering to the patientcyclophosphamide at a dose between 200 mg/m² and 2000 mg/m², e.g., 200mg/m², 300 mg/m², 500 mg/m², 400 mg/m², 600 mg/m², 700 mg/m², 800 mg/m²,900 mg/m², 1000 mg/m², or 1110 mg/m², and fludarabine at a dose between20 mg/m² and 900 mg/m², e.g., 20 mg/m², 25 mg/m², 30 mg/m², 35 mg/m², 40mg/m², 45 mg/m², or 50 mg/m², wherein the patient is treated with a Tcell therapy after exhibiting an increased serum level of IL-15, IP-10,and/or IL-7 and/or a decreased serum level of perforin. In otherembodiments, the method of the invention is to identify a patientsuitable for a T cell therapy comprising (i) administering to thepatient cyclophosphamide at a dose between 200 mg/m² and 2000 mg/m²,e.g., 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m²,800 mg/m², 900 mg/m², 1000 mg/m², or 1110 mg/m², and fludarabine at adose between 20 mg/m² and 900 mg/m², e.g., 20 mg/m², 25 mg/m², 30 mg/m²,35 mg/m², 40 mg/m², 45 mg/m², or 50 mg/m², and (ii) administering a Tcell therapy after the patient exhibits an increased serum level ofIL-15, IP-10, and/or IL-7 and/or a decreased serum level of perforin. Inother embodiments, the invention is directed to a method for identifyinga patient suitable for a T cell therapy comprising (i) administering tothe patient cyclophosphamide at a dose between 200 mg/m² and 2000 mg/m²,e.g., 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m²,800 mg/m², 900 mg/m², 1000 mg/m², or 1110 mg/m², and fludarabine at adose between 20 mg/m² and 900 mg/m², e.g., 20 mg/m², 25 mg/m², 30 mg/m²,35 mg/m², 40 mg/m², 45 mg/m², or 50 mg/m², (ii) administering anadditional amount of cyclophosphamide or fludarabine or administering aneffective amount of IL-15, IP-10, and/or IL-7, when the patient does notexhibit a sufficient serum level of IL-15, IP-10, and/or IL-7, and (iii)administering a T cell therapy after the patient exhibits an increasedserum level of IL-15, IP-10, and/or IL-7. In certain embodiments, the Tcell therapy is administered to the patient when the patient exhibits anincreased serum level of at least one additional cytokine selected fromthe group consisting of MCP-1, CRP, PLGF, IP-10, and any combinationthereof.

The methods of the invention further comprise measuring the serum levelof IL-15, IP10, perforin, and/or IL-7. In one embodiment, the serumlevel of IL-7 in the patient is increased at least 2 fold, at least 3fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 15fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 60fold, at least 70 fold, at least 80 fold, or at least 90 fold after theadministration compared to the IL-7 serum level prior to theadministration of cyclophosphamide and fludarabine. In anotherembodiment, the serum level of IL-15 in the patient is increased atleast 5 fold, at least 10 fold, at least 15 fold, at least 20 fold, atleast 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, atleast 45 fold, at least 50 fold, at least 60 fold, at least 70 fold, atleast 80 fold, or at least 90 fold after the administration compared tothe IL-15 serum level prior to the administration of cyclophosphamideand fludarabine.

In other embodiments, the serum level of MCP-1 in the patient that isincreased after the administration of cyclophosphamide at a dose between200 mg/m² and 2000 mg/m², e.g., 200 mg/m², 300 mg/m², 400 mg/m², 500mg/m², 600 mg/m², 700 mg/m², 800 mg/m², 900 mg/m², 1000 mg/m², or 1110mg/m², and fludarabine at a dose between 20 mg/m² and 900 mg/m², e.g.,20 mg/m², 25 mg/m², 30 mg/m², 35 mg/m², 40 mg/m², 45 mg/m², or 50 mg/m²,is increased by at least 1.5 fold, at least 2 fold, at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, or atleast 20 fold compared to the MCP-1 serum level prior to theadministration of cyclophosphamide and fludarabine. In some embodiments,the serum level of PLGF in the patient is increased at least 1.5 fold,at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, atleast 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, atleast 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, atleast 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, atleast 90 fold, or at least 100 fold after the administration compared tothe PLGF serum level prior to the administration of cyclophosphamide andfludarabine. In certain embodiments, the serum level of CRP in thepatient is increased at least 1.5 fold, at least 2 fold, at least 3fold, at least 4 fold, at least 5 fold, at least about 9 fold, at least10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least90 fold, or at least 100 fold after the administration compared to theCRP serum level prior to the administration of cyclophosphamide andfludarabine. In yet other embodiments, the serum level of IP-10 in thepatient is increased at least 2 fold, at least 3 fold, at least 4 fold,at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, orat least 30 fold after the administration compared to the IP-10 serumlevel prior to the administration of cyclophosphamide and fludarabine.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allreferences cited throughout this application are expressly incorporatedherein by reference.

EXAMPLES Example 1

A phase ½, single arm, open label, trial was designed to determine thesafety and feasibility of anti-CD19 CAR+ T cells administered tosubjects with B cell malignancies.

Subjects who signed informed consent and met study eligibility wereenrolled into the study and underwent leukapheresis to obtain PBMCs forthe production of anti-CD19 CAR+ T cells. Subjects were treated withconditioning chemotherapy prior to hospitalization in preparation for asingle infusion of anti-CD19 CAR+ T cells on Day 0. Some subjects werethen treated with interleukin-2 (Group 1 only), 3 hours after theanti-CD19 CAR+ T cell infusion. Retreatment of a second dose ofanti-CD19 CAR+ T cells was allowed if there was a response of partialresponse (PR) or complete response (CR) after the first infusion andthen subsequent disease progression.

Three groups of subjects were enrolled. Group 1 includes 8 subjects,including 1 subject who was retreated, dosed with anti-CD19 CAR+ T cellsranging from 3×10⁶ through 30×10⁶ anti-CD19 CAR+ T cells/kg. The dose ofanti-CD19 CAR+ T cells followed a conditioning regimen consisting ofhigh dose cyclophosphamide at 60-120 mg/kg (2220-4440 mg/m²) for twodays followed by fludarabine at 25 mg/m² for five days. These subjectsalso received high dose interleukin-2 (IL-2) at 720,000 IU/kg (every 8hours until 15 doses or toxicity precluded additional doses) after theanti-CD19 CAR+ T cell administration to stimulate their proliferation.

Group 2 includes 15 subjects, including 2 subjects from Group 1 who wereretreated, who received high dose cyclophosphamide and fludarabine andno interleukin-2 following varying doses of anti-CD19 CAR+ T celladministration (1×10⁶ through 5×10⁶ anti-CD19 CAR+ T cells/kg).

Group 3 includes 11 subjects, who have received a reduced conditioningregimen of cyclophosphamide at 300 mg/m² and fludarabine at 30 mg/m²,both given for 3 concurrent days with no IL-2. The first 7 and last 4 ofthese subjects received an anti-CD19 CAR+ T cell infusion of 1×10⁶anti-CD19 CAR+ T cells and 2×10⁶ anti-CD19 CAR+ T cells, respectively.

Demographics

Subject demographic and disease characteristics are provided in Table 1.Thirty-two (32) subjects were enrolled, 19 subjects (59%) had DLBCL orPMBCL, 7 subjects (22%) had CLL, and 6 subjects (19%) had other indolentNHL, including indolent follicular lymphoma and splenic marginal zonelymphoma. Most subjects had refractory disease (84%), and had received amedian of 3 prior lines of therapy. All subjects with aggressive NHLreceived prior anti-CD20 therapy, platinum combination chemotherapy, and95% received prior anthracycline-based chemotherapy.

Pharmacokinetics

The number of anti-CD19 CAR+ T cells in the peripheral blood at varioustime points after initial administration on Day 0 were evaluated usingqPCR analysis and corroborated by standard curves generated by flowcytometry with an antibody reagent specific for scFv present in theanti-CD19 CAR construct (Kochenderfer et al., “B-cell depletion andremissions of malignancy along with cytokine-associated toxicity in aclinical trial of anti-CD19 chimeric-antigen-receptor-transduced Tcells,” Blood 119:2709-20 (2012)).

In group 1, 3×10⁶ to 30×10⁶ anti-CD19 CAR+ T cells/kg were infused. Inthe first 6 subjects, the anti-CD19 CAR+ T cells in blood circulationwere detected at higher levels within 2 weeks after infusion, reachingup to 0.02-1% of total PBMC, then decayed rapidly and were undetectableafter 50 days. Subjects 7 and 8, dosed with the highest number ofanti-CD19 CAR+ T cells (28 and 30×10⁶ anti-CD19 CAR+ T cells/kg,respectively), had higher peak percentages reaching >10% anti-CD19 CAR+T cells of total PBMC, and longer-term persistence of anti-CD19 CAR+ Tcells in blood (>130 and 180 days, respectively).

TABLE 1 Demographics of clinical trial subjects. Group 1 Group 2 Group 3Total (N = 8) (N = 15) (N = 11) (N = 32) Age (years) Mean (std) 56 (6)52 (11) 50 (16) 52 (12) Median 56 55 55 55 Minimum, 47, 63 31, 69 29, 6729, 69 maximum Gender Male 8 (100%) 8 (53%) 11 (100%) 25 (78%) Female 0(0%) 7 (47%) 0 (0%) 7 (22%) Race White 8 (100%) 13 (87%) 10 (91%) 29(91%) Asian 0 (0%) 1 (7%) 0 (0%) 1 (3%) Black or 0 (0%) 1 (7%) 0 (0%) 1(3%) African American Unknown 0 (0%) 0 (0%) 1 (9%) 1 (3%) Diagnosis CLLFL 4 (50%) 4 (27%) 0 (0%) 7 (22%) SMZL 3 (38%) 0 (0%) 1 (9%) 4 (13%)iNHL 1 (13%) 1 (7%) 0 (0%) 1 (3%) DLBCL 0 (0%) 1 (7%) 0 (0%) 1 (3%)PMBCL 0 (0%) 5 (33%) 8 (73%) 13 (41%) 0 (0%) 4 (27%) 2 (18%) 6 (19%)Prior 7 (88%) 13 (87%) 11 (100%) 30 (94%) anti-CD20 Refractory to lastline of therapy (SD/PD to last line) Yes 6 (75%) 13 (87%) 9 (82%) 27(84%) No 1 (13%) 2 (13%) 0 (0%) 2 (6%) Unknown 1 (13%) 0 (0%) 2 (18%) 3(9%) Lines of prior therapy Median 4 (2, 7) 3 (1, 12) 3 (2, 10) 3 (1,12) (minimum, maximum)

In group 2, in the absence of interleukin-2 treatment, the anti-CD19CAR+ T cells showed a similar expansion in the peripheral blood within 2weeks, followed by decay and complete disappearance from circulationwithin several weeks (Table 2).

Overall, there was no overt relationship between the dose of anti-CD19CAR+ T cells and their expansion and persistence in the peripheralblood. Likewise, to date, there was no apparent relationship between theanti-CD19 CAR+ T cell dose, the anti-CD19 CAR+ T cell expansion orpersistence in the blood, and the clinical response or the toxicitiesrelated to this therapy, respectively.

TABLE 2 Anti-CD19 CAR+ T cell expansion and persistence in theperipheral blood of subjects in group 2. Dose range of Total dose ofanti-CD19 Anti-CD19 anti-CD19 CAR+ T CAR+ T cell Persistence CAR+ Tcells/kg in peak - expressed Time of anti-CD19 cells millions as numberof to peak CAR+ T cells (×10⁶) (×10⁶) cells/μL blood in days in daysMean 210 3.1 50 10 32 (Range) (105-490) (1.2-7.5) (9-777) (7-17)(13-132)

In groups 1 and 2, there was no secondary expansion of anti-CD19 CAR+ Tcells following their primary expansion at 7-14 days post-infusion.There is no evidence of oncogenic transformation ascribable to thegenomic insertion of the CAR-expression retrovirus in the subjectstested to date. Group 3 results were not yet available at the time ofdata cutoff.

Efficacy

Clinicians evaluated 32 subjects for safety and 29 subjects forefficacy. The overall response rate for the 29 subjects evaluable forefficacy was 76%. Eleven (11) of 29 subjects (38%) achieved a CR and11/29 subjects (38%) achieved a PR (FIGS. 2A and 2B; Table 3).

Sixteen of the 29 (55%) evaluable subjects remain in response from theirfirst treatment, with 12 subject's (including retreated subjects)duration of response exceeding 1 year (Table 3). Three respondingsubjects were retreated after progression, all have ongoing responses(17.4 to over 52.2 months).

As indicated in Table 3, 17 of the 19 subjects with refractoryaggressive DLBCL/PMBCL were evaluable for disease response (1 subjectwas not evaluable; 1 subject had not yet been evaluated). Among these 17subjects, 11 (65%) had a response with 6/17 subjects (35%) achieving aCR. The median duration of response is 7.3 months.

Six of the 7 evaluable subjects (86%) with CLL had a response with 4/7subjects (57%) achieving a CR (Table 3). The median duration of responseis 22.2 months with 4/7 subjects (57%) still in response including 3subjects with ongoing responses for greater than 27 months (Table 3).

TABLE 3 Objective Response Rate and Duration of Response by Tumor Type.Overall Complete Duration of Response Tumor Type Response Response(months) Median (n evaluable) Rate n (%) Rate n (%) (Individual) Any (n= 29) 22 (76%) 11 (38%) 14.9 DLBCL/PMBCL 11 (65%) 6 (35%)  7.3 (n = 17)(<1+, 1.0, 1.2, 5.3+, 6.0, 7.3, 7.9+, 14.1+, 15.7+, 20.3+, 28.5+) CLL 6(86%) 4 (57%) 22.2 (n = 7) (2.8, 4.6, 17.1+, 27.2+, 31.1+, 35.6+)Indolent NHL 5 (100%) 1 (20%) 18.8 (n = 5) (10.4+, 17.1+, 18.8+, 45.4+,58.5+) +indicates that the response is still ongoing

Five of the 5 evaluable subjects (100%) with indolent NHL had a responsewith 1/5 subjects (20%) achieving a CR. The median duration of responseis 18.8 months (Table 3). Five subjects (5/5; 100%) remain in responsewith 2 subjects responding greater than 45 months (Table 4).

Safety

Adverse Events

32 subjects had been treated with the anti-CD19 CAR+ T cells with noadverse events yet reported for the last subject treated. Overall safetysummaries include all 32 treated subjects. Summaries by group includesafety data for subjects 1010003 and 1010004 twice, once when thesesubjects were treated in Group 1 and second when these subjects weretreated in Group 2 (retreatment with anti-CD19 CAR+ T cells).

Summary of Adverse Events

A summary of adverse events is provided in Table 4. Overall, 31 subjects(97%) experienced any adverse event, with 0 subjects (0%) experiencing aworst grade of grade 3, 29 subjects (91%) experiencing a worst grade ofgrade 4, and 2 subjects (6%) with fatal adverse events. Twenty subjects(63%) experienced an anti-CD19 CAR+ T cell related adverse event; 6subjects (19%) worst grade of 3, 8 subjects (25%) worst grade 4, and nosubjects experienced a grade 5 event. Sixteen (16) subjects (50%)experienced a serious adverse event; 3 subjects (9%) worst grade of 3, 9subjects (28%) worst grade of 4, and 2 subjects (6%) worst grade of 5.

TABLE 4 Summary of Adverse Events. Group 1 Group 2 Group 3 Overall n (%)(N = 8) (N = 15) (N = 11) (N = 32) Any Gr 2-5 AE 8 (100) 15 (100%) 10(91%) 31 (97%) Gr 3 0 (0) 0 (0%) 0 (0%) 0 (0%) Gr 4 7 (88%) 14 (93%) 10(91%) 29 (91%) Gr 5 1 (13%) 1 (7%) 0 (0%) 2 (6%) Any Gr 2-5 3 (37%) 11(73%) 7 (64%) 20 (63%) CAR related Gr 3 0 (0%) 3 (20%) 3 (27%) 6 (19%)Gr 4 2 (25%) 6 (40%) 0 (0%) 8 (25%) Gr 5 0 (0) 0 (0%) 0 (0%) 0 (0%) AnySerious 6 (75%) 8 (53%) 2 (18%) 16 (50%) Gr 3 2 (25%) 1 (7%) 0 (0%) 3(9%) Gr 4 2 (25%) 6 (40%) 1 (9%) 9 (28%) Gr 5 1 (13%) 1 (7%) 0 (0%) 2(6%)Dose-Limiting Toxicity

The incidence of DLT within Groups 1, 2 and 3 was 38%, 40%, and 0%,respectively. With the exception of subject 1010002, DLTs were primarilyneurotoxicities, 2 cases of elevated creatinine, and 1 event each ofhypoxia and hypotension. Table 6 provides a listing of DLTs. In Group 3there were no DLTs reported. The conditioning regimen in Group 3 wasstudied with 2×10⁶ anti-CD19 CAR+ T cells/kg.

TABLE 5 Dose-Limiting Toxicities. Anti-CD19 Subject CAR+ T Dose-LimitingNo. cells/kg Toxicities (DLT) Group Comment 1010002 3 × 10⁶ G4 hypoxia 1The subject G4 influenza had a infection culture- G5 thrombosis provenH1N1 (cerebral thrombi viral with global pneumonia infarction) and died18 days after his infusion. 1010004 2.5 × 10⁶  G4 creatinine 2 Requireddialysis 1010007 28 × 10⁶  G4 somnolence 1 Required intubation 101000830 × 10⁶  G4 somnolence 1 Required intubation 1010009 5 × 10⁶ G3confusion/ 2 aphasia G3 cranial nerve VII neuropathy 1010010 4 × 10⁶ G3intermittent 2 confusion/aphasia 1010014 2.5 × 10⁶  G3 hypoxia 2Required G4 hypotension intubation G3 creatinine G4 somnolence/intermittent confusion 1010015 2.5 × 10⁶  G4 myoclonus 2 Required G4expressive intubation aphasia 1010021 1 × 10⁶ G4 aphasia 2 G3 motorneuropathyCytokine Release Syndrome

Cytokine release is induced by the activated T cells upon engagementwith the CD19 target. Using a broad search strategy, treatment-emergentadverse events which may be attributed to CRS include fever, febrileneutropenia, hypotension, acute vascular leak syndrome, elevatedcreatinine, renal failure, hypoxia, and pleural effusion. Twenty-eight(28) (88%) subjects reported adverse events which could be attributed tocytokine release, where 24 subjects (75%) reported a ≧grade 3 event and6 subjects (19%) experienced a serious event. Adverse events due toco-therapies such as IL-2 (used in Group 1) and conditioningchemotherapy (causing febrile neutropenia) potentially confound thisanalysis.

Clinical manifestations of CRS occurred typically in the first weekafter anti-CD19 CAR+ T cell infusion and were less common in thesubjects in Group 3. Only 1 of the 11 subjects in Group 3 experiencedgrade 3 hypotension, and 4 experienced grade 3 fever. Events of acutevascular leak syndrome, oliguria, elevated creatinine, and renal failurewere reported only in subjects in Groups 1 and 2.

Neurologic Adverse Events

Neurologic adverse events were observed in all three groups,predominantly aphasia/dysphasia, confusion, motor neuropathy andsomnolence. Thirteen subjects (41%) had severe ≧grade 3 neurotoxicity,and 11 subjects (34%) experienced a serious event.

The subject who died with a neurotoxicity had an event of CNScerebrovascular ischemia in the context of viral influenza A infection.This was deemed unrelated to the anti-CD19 CAR+ T cells by theinvestigator.

Five subjects (16%) with neurotoxicity events required mechanicalventilation for airway protection for neurological adverse events; allof these subjects were in Groups 1 and 2. There have been no subjectsintubated in Group 3.

Neurologic adverse events had a median onset of 6 days ranging betweendays 2 and 17 post anti-CD19 CAR+ T cell infusion, with the exception ofgrade 4 myelitis which occurred in 1 subject and had an onset at day 110post anti-CD19 CAR+ T cell infusion. Given the time of onset,presentation and brain MRI findings, this event was considered by theinvestigator to be related to fludarabine and not attributed to theanti-CD19 CAR+ T cells. The median time to resolution of theneurological adverse event to grade 1 or better was 14 days postinfusion.

Deaths

Two subjects died within 30 days of chemotherapy and anti-CD19 CAR+ Tcell infusion. Subject 2 died 18 days after investigational treatmentdue to a cerebral infarction concurrent with viral pneumonia, influenzaA infection, E. coli infection, dyspnea, and hypoxia. Subject 11 hadPMBCL, with extensive fibrotic mediastinal lymphoma involvement, died 16days after investigational treatment. No cause of death determined onautopsy and the autopsy report concluded likely cause of death wascardiac arrhythmia given the mediastinal involvement of PMBCL. Neitherevent was deemed related to anti-CD19 CAR+ T cells by the investigator.

Example 2

Select patients were administered a conditioning chemotherapy comprisingcyclophosphamide 300 mg/m²/day and Fludarabine 30 mg/m²/day. Theconditioning chemotherapy was administered for three days from day −5 today −3. On day 0, a first subset of the patients (patients 22-28) (Table6) received 10 day-manufacturing, fresh anti-CD19 CAR+ T cells, and asecond subset of the patients (patients 29-32) received 6day-manufacturing, cryopreserved anti-CD19 CAR+ T cells.

TABLE 6 Condition and Outcome Data for Patients 22-28. Patient ConditionOutcome 22 DLBCL PR 23 FL PR 24 DLBCL PR 25 DLBCL PR 26 DLBCL PD 27DLBCL CR 28 DLBCL PD DLBCL = Diffuse Large B Cell Lymphoma; FL =Follicular Lymphoma; PR = Partial Response; CR = Complete Response; PD =Progressive Disease

Patient sera was tested by luminex using Millipore HCD8MAG15K17PMX kit(T1, T2, immune modulating cytokines, chemokines, immune effectors). Thelevels of interleukin 15 (IL-15), monocyte chemotactic protein 1(MCP-1), gamma-induced protein 10 (IP-10), placental growth factor(PLGF), soluble intercellular adhesion molecule 1 (sICAM-1), C-reactiveprotein (CRP), vascular endothelial growth factor D (VEGF-D), macrophageinflammatory protein 1β (MIP-1β) were measured before and afterconditioning.

Of patients 22-28, patients 22-25 and 27 showed at least a partialresponse and patients 26 and 28 showed progressive disease followingtreatment. For patients 22-26, the levels of IL-15, MCP-1, and PLGFshowed at least some increase in patient sera (FIGS. 4A, 4B, and 4D),while the levels of IP-10, sICAM-1, CRP, VEGF-D, and MIP-1β increased insome patients and remained stable or decreased in others (FIGS. 4C and4E-4H). Only IL-15 was measured for patients 27 and 28 (FIG. 4A).

Some differences in marker levels were observed between respondingpatients, having either a partial or complete response, andnon-responding patients, having progressive disease. IL-15 levelsincreased by an average of about 35-Fold in responding patients, rangingfrom about 10-fold to about 55-fold, relative to baseline, while thenon-responding patients each had a less than about 10-fold increase inIL-15 levels (FIG. 5A). MCP-1 levels in responders increased by anaverage of about 5 fold, ranging from about 2 fold to about 7 fold,while the non-responder (patient 26) had a less than 4-fold increase inthe level of MCP-1 (FIG. 5B). IP-10 levels in responders increased by anaverage of about 3.5 fold, ranging from about 2 fold to about 7 fold,while the non-responder had essentially no change in the level of serumIP-10 (FIG. 5C). PLGF levels in responders increased by an average ofabout 30 fold, ranging from a slight increase of about 2 fold or less toan increase of about over 100 fold, while the non-responder had only aslight increase in the level of serum PLGF (FIG. 5D). sICAM-1 levels inresponders increased by an average of about 3 fold, ranging fromessentially no change to an increase of about 4.5 fold, while thenon-responder had essentially no change in the level of serum sICAM-1(FIG. 5E). CRP levels in responders increased by an average of about 10fold, ranging from essentially no change to an increase of about 25fold, while the non-responder had essentially no change in the level ofserum CRP (FIG. 5F). VEGF-D levels in responders increased by an averageof about 3 fold, ranging from essentially no change to an increase ofabout 6 fold, while the non-responder had essentially no change in thelevel of serum VEGF-D (FIG. 5G). MIP-1β levels in responders increasedby an average of about 1.5 fold, ranging from essentially no change toan increase of about 3 fold, while the level of serum MIP-1β decreasedby about 50% in the non-responder (FIG. 5H).

Patients 30-33 were dosed with 6 day-manufacturing, cryopreserved cells,and the levels of various cytokines, chemokines, effectors, markers ofinflammation, and adhesion molecules, including granulocyte macrophagecolony-stimulating factor (GM-CSF), interferon γ (IFNγ or IFNG),interleukin 10 (IL-10), IL-15, interleukin 2 (IL-2), interleukin 5(IL-5), interleukin 6 (IL-6), interleukin 8 (IL-8), IP-10, MCP-1,MIP-1β, serum granzyme A (GRNZA), serum granzyme B (GRNZB), PLGF, CRP,monocyte chemotactic protein 4 (MCP-4), interleukin 16 (IL-16), thymusand activation regulated chemokine (TARC), Eotaxin-3, sICAM-1, solublevascular adhesion molecule 1 (sVCAM-1), and serum amyloid A (SAA), weremeasured on selected days from day −6 through day 18 (FIGS. 6A-6V).

Example 3

To improve the depth and duration of lymphocyte depletion observed ingroup 3 of Example 1, the conditioning chemotherapy dose in cohort A1will be increased to cyclophosphamide at 500 mg/m² and fludarabine at 30mg/m² both given for 3 concurrent days with the target dose of 2×10⁶anti-CD19 CAR+ T cells/kg. The cyclophosphamide dose used in thisregimen (Cohort A1) is approximately 38% lower than that used in theGroup 2 cyclophosphamide 30 mg/kg conditioning regimen from Example 1(incidence of dose limiting toxicity (DLT) 29%), with the same lowerdose of fludarabine dose as Group 3 of Example 1.

Evaluation of higher conditioning chemotherapy doses and/or varyinganti-CD19 CAR+ T cell doses would proceed based on the incidence of DLTand evaluation of benefit-risk. The CAR vector construct is identical tothe construct described in Example 1. This example describes a clinicaltrial designed to test the safety and efficacy of anti-CD19 CAR+ T cellsgenerated by a rapid, closed, and bead-less process. Closing the processretains the characteristics of the T cell product.

Study Design

A phase ½ multicenter, open-label study will be performed evaluating thesafety and efficacy of KTE-C19 in subjects with refractory NHL. Thestudy will be separated into two distinct phases designated as phase 1and phase 2.

During phase 1, approximately 6 to 24 subjects with DLBCL, PMBCL or TFLwill be enrolled to evaluate the safety of KTE-C19 regimens. A safetyreview team (SRT), internal to the study sponsor, will review the safetydata and make recommendations on further study conduct of phase 1 andprogression to phase 2 as depicted in FIG. 3.

During phase 2, subjects will enroll into two separate cohortsdesignated as cohort 1 and cohort 2. Cohort 1 will enroll adult subjectswith refractory DLBCL, and cohort 2 will enroll adult subjects withrefractory PMBCL and TFL. TFL is defined as subjects who received priorchemotherapy for follicular lymphoma.

Independent of the phase of the study each subject will follow the samestudy treatment schedule and procedural requirements. Each subject willproceed through the following study periods: screening/leukapheresisperiod; conditioning chemotherapy period; investigational product (IP)treatment period; post treatment assessment period Long-term follow-upperiod

Study Duration

For an individual subject, the length of participation includes an up to28-day screening period, a 5-7 day conditioning chemotherapy treatmentperiod, a KTE-C19 treatment period (which includes a 7-day in-hospitalrecovery period), a post treatment assessment period, and a long termfollow-up period (survival surveillance for up to 15 years).

Subjects will be followed for all adverse events for 3 months aftertreatment. After 3 months, subjects will be monitored for targetedadverse events/serious adverse events (e.g., hematological,neurological, second malignancies, infections or autoimmune disorders)and presence of replication competent retrovirus (RCR) in subjects bloodat intervals outlined in the schedule of assessments (SOA). The need forprolonged follow-up is based on the potential persistence of genetransfer vectors in treated subjects.

Completion of the study is defined as the time at which the last subjectcompletes the long term follow-up period visit, is considered lost tofollow-up, withdraws consent, or dies. The primary analyses will beconducted when all subjects in cohort 1 of phase 2 and the overall studypopulation, respectively have completed the 6 month disease responseassessment, are lost to follow-up, withdraw from the study, or die,whichever occurs first.

Subject Eligibility

The inclusion criteria for subjects include:

-   a) Histologically confirmed aggressive B cell NHL, including the    following types defined by WHO 2008: DLBCL not otherwise specified,    T cell/histiocyte rich large B cell lymphoma, DLBCL associated with    chronic inflammation, Epstein-Barr virus (EBV)+ DLBCL of the    elderly; primary mediastinal (thymic) large B cell lymphoma; or    transformation of follicular lymphoma to DLBCL;-   b) Chemotherapy-refractory disease, defined as one or more of stable    disease (duration of stable disease must be ≦12 months) or    progressive disease as best response to most recent chemotherapy    containing regimen; and disease progression or recurrence ≦12 months    of prior autologous SCT;-   c) subjects must have received adequate prior therapy including at a    minimum anti-CD20 monoclonal antibody unless investigator determines    that tumor is CD20 negative and an anthracycline containing    chemotherapy regimen;-   d) Subjects with transformed FL must have received prior    chemotherapy for follicular lymphoma and subsequently have    chemorefractory disease after transformation to DLBCL;-   e) At least 1 measurable lesion according to the revised IWG    Response Criteria for Malignant Lymphoma; lesions that have been    previously irradiated will be considered measurable only if    progression has been documented following completion of radiation    therapy;-   f) MRI of the brain showing no evidence of central nervous system    lymphoma;-   g) Greater than or equal to 2 weeks must have elapsed since any    prior radiation therapy or systemic therapy at the time the subject    is planned for leukapheresis;-   h) Toxicities due to prior therapy must be stable or recovered to    ≦Grade 1 (except for clinically non-significant toxicities such as    alopecia);-   i) Subjects must be age 18 or older;-   j) Eastern cooperative oncology group (ECOG) performance status of 0    or 1-   k) Subjects must have the following laboratory values: i)    ANC≧1000/uL; ii) Platelet count ≧50,000/uL; iii) Adequate renal,    hepatic, and cardiac function defined as serum creatinine ≦1.5    mg/dL, serum ALT/AST≦2.5 ULN, and total bilirubin ≦1.5 mg/dl, except    in subjects with Gilbert's syndrome; and iv) Cardiac ejection    fraction ≧50% and no evidence of pericardial effusion as determined    by an ECHO; and-   l) Females of childbearing potential must have a negative serum or    urine pregnancy test.

The exclusion criteria for subjects includes:

-   a) History of malignancy other than nonmelanoma skin cancer or    carcinoma in situ (e.g., cervix, bladder, breast) or follicular    lymphoma unless disease free for at least 3 years;-   b) History of Richter's transformation of CLL;-   c) Autologous stem cell transplant within 6 weeks of informed    consent;-   d) History of allogeneic stem cell transplantation;-   e) Prior CD19 targeted therapy with the exception of subjects who    received KTE-C19 in this study and are eligible for re-treatment;-   f) Prior chimeric antigen receptor therapy or other genetically    modified T cell therapy;-   g) History of severe, immediate hypersensitivity reaction attributed    to aminoglycosides;-   h) Clinically significant active infection (e.g., simple UTI,    bacterial pharyngitis allowed) or currently receiving IV antibiotics    or have received IV antibiotics within 7 days prior to enrollment    (Prophylaxis antibiotics, antivirals and antifungals are permitted);-   i) Known history of infection with HIV or hepatitis B (HBsAg    positive) or hepatitis C virus (anti-HCV positive);-   j) Subjects with detectable cerebrospinal fluid malignant cells, or    brain metastases, or with a history of cerebrospinal fluid malignant    cells or brain metastases;-   k) History of a seizure disorder, cerebrovascular    ischemia/hemorrhage, dementia, cerebellar disease, or any autoimmune    disease with CNS involvement;-   l) Subjects with cardiac atrial or cardiac ventricular lymphoma    involvement;-   m) Requirement for urgent therapy due to tumor mass effects such as    bowel obstruction or blood vessel compression;-   n) Primary immunodeficiency;-   o) Any medical condition likely to interfere with assessment of    safety or efficacy of study treatment;-   p) Current or expected need for systemic corticosteroid therapy;    topical and inhaled corticosteroids in standard doses and    physiologic replacement for subjects with adrenal insufficiency are    allowed; doses of corticosteroids of greater than or equal to 5    mg/day of prednisone or equivalent doses of other corticosteroids    are not allowed;-   q) History of severe immediate hypersensitivity reaction to any of    the agents used in this study;-   r) Live vaccine ≦6 weeks prior to start of conditioning regimen;-   s) Women of child-bearing potential who are pregnant or    breastfeeding, because of the potentially dangerous effects of the    preparative chemotherapy on the fetus or infant; females who have    undergone surgical sterilization or who have been postmenopausal for    at least 2 years are not considered to be of childbearing potential;-   t) Subjects of both genders who are not willing to practice birth    control from the time of consent through 6 months after the    completion of KTE-C19; and-   u) In the investigators judgment, the subject is unlikely to    complete all protocol-required study visits or procedures, including    follow-up visits, or comply with the study requirements for    participation.

In addition, biomarker analysis will be performed on blood and tumorsamples to evaluate predictive and pharmacodynamic markers for KTE-C19.Prognostic markers in aggressive NHL may also be evaluated. Baselineleukapheresis and final KTE-C19 samples will be banked and may beanalyzed by immunophenotyping and/or gene expression profiling.Remaining samples may be stored for future exploratory analysis of DNA,RNA, or protein markers. Archived tumor tissue will be collected forcentral path review. Additional analysis may include CD19 expression,gene expression profiling, and analysis of DNA alterations. Remainingtumor samples may be stored for future exploratory analysis of DNA, RNA,or protein markers.

Protocol Treatment

Schedule

Leukocytes will be obtained from subjects by leukapheresis (12-15 literapheresis with a goal to target approximately 5-10×10⁹ mononuclear cellsfor the manufacturing of KTE-C19. Each subject's leukapheresed productwill be processed to enrich for the T cells containing PBMC fraction. Tcells are then stimulated to expand and transduced with a retroviralvector to introduce the CAR gene. The T cells are then expanded andcryopreserved to generate the investigational product. Followingcompletion of each subject's conditioning chemotherapy regimen, subjectswill receive their respective KTE-C19 infusion.

Study Treatment

Subjects will receive a non-myeloablative conditioning regimenconsisting of cyclophosphamide and fludarabine in order to inducelymphocyte depletion and create an optimal environment for expansion ofKTE-C19 in vivo. Subjects will initiate conditioning chemotherapy withcyclophosphamide and fludarabine beginning on Day −5 (or Day −7 forcohort B) through Day −1. The 5-day conditioning chemotherapy regimenwill be administered in an outpatient setting. The 7-day conditioningchemotherapy regimen may be administered as an outpatient or inpatientregimen per investigator's discretion.

Phase 1:

In Cohorts A1 and A2, subjects will receive the following 5-dayconditioning chemotherapy regimen: IV hydration with 1 L of 0.9% NaClsaline solution given prior to cyclophosphamide on the day of infusion;followed by Cyclophosphamide 500 mg/m² IV over 60 minutes on Day −5, Day−4, and Day −3; followed by Fludarabine 30 mg/m² IV over 30 minutes onDay −5, Day −4, and Day −3; followed by an additional 1 L of 0.9% NaClsaline solution at the completion of the fludarabine infusion (FIG. 3).In certain cases, mesna (sodium 2-mercaptoethanesulfonate) can be addedper institutional guidelines.

In Cohort A3, subjects will receive the following 5-day chemotherapyregimen: IV hydration with 1 L of 0.9% NaCl saline solution given priorto cyclophosphamide on the day of infusion; followed by Cyclophosphamide300 mg/m² IV over 60 minutes on Day −5, Day −4, and Day −3; followed byFludarabine 30 mg/m² IV over 30 minutes on Day −5, Day −4, and Day −3;followed by an additional 1 L of 0.9% NaCl saline solution at thecompletion of the fludarabine infusion. In certain cases, mesna may beadded per institutional guidelines

For subjects enrolled into Cohorts A1, A2, or A3, Day −2 and Day −1 willbe rest days before KTE-C19 infusion on Day 0.

In Cohorts B1 and B2, subjects will receive the following 7-daychemotherapy regimen: IV hydration with 0.9% NaCl saline solution,recommended at 2.6 ml/kg/hr (maximum 200 ml/hr), administered as acontinuous infusion starting 11 hours pre-cyclophosphamide infusion andcontinue hydration until 24 hours after last cyclophosphamide infusion;Cyclophosphamide 30 mg/kg (1110 mg/m²) IV administered on Day −7 and −6,infused over 120 minutes; followed by Fludarabine 25 mg/m² IVadministered on Day −5, Day −4, Day −3, Day −2 and Day −1, infused over30 minutes. In certain cases, mesna may be added per institutionalguidelines.

For subjects enrolled into Cohort B1 or B2, there will be no rest daysbetween the last day of chemotherapy (Day −1) and the KTE-C19 infusionon Day 0.

For KTE-C19, subjects in Cohorts A1, A3, or B1 will receive KTE-C19treatment consisting of a single infusion of CAR transduced autologous Tcells administered intravenously at a target dose of 2×10⁶ anti-CD19CAR+ T cells/kg (±20%; 1.6×10⁶ anti-CD19 CAR+ T cells/kg to 2.4×10⁶anti-CD19 CAR+ T cells/kg). A minimum dose of 1×10⁶ anti-CD19 CAR+ Tcells/kg may be administered. For subjects weighing greater than 100 kg,a maximum flat dose of 2×10⁸ anti-CD19 CAR+ T cells will beadministered.

Subjects in Cohorts A2 or B2 will receive KTE-C19 treatment consistingof a single infusion of CAR transduced autologous T cells administeredintravenously at a target dose of 1×10⁶ anti-CD19 CAR+ T cells/kg (±20%;0.8×10⁶ anti-CD19 CAR+ T cells/kg to 1.2×10⁶ anti-CD19 CAR+ T cells/kg).A minimum dose of 0.5×10⁶ anti-CD19 CAR+ T cells/kg may be administered.For subjects weighing greater than 100 kg, a maximum flat dose of either1×10⁸ anti-CD19 CAR+ T cells will be administered.

Phase 2:

A KTE-C19 regimen determined by the SRT to be safe in phase 1 will becarried forward into the phase 2 portion of the study.

Retreatment

Subjects who achieved a PR or CR can receive a second course ofconditioning chemotherapy and KTE-C19 if their disease subsequentlyprogresses (and the relapse is not known to be CD19-malignant cells). Tobe eligible for a second course of treatment, subjects should bere-evaluated and continue to meet the original study eligibilitycriteria, with the exception of exclusion criteria related to prior CARtherapy, and should not have received subsequent chemotherapy for thetreatment of lymphoma. Furthermore, any toxicity related to fludarabineor cyclophosphamide should be stable and resolved to less than grade 1prior to retreatment with the exception of alopecia. A maximum of 1retreatment course may occur per subject. Subjects enrolled in phase 2will receive the same KTE-C19 regimen. Subjects enrolled in phase 1 willreceive the KTE-C19 regimen selected for phase 2. If the phase 2 regimenhas not yet been selected, subjects will receive the last KTE-C19regimen that was determined safe by the SRT.

Subjects who experience a DLT in phase 1 or a comparable toxicity inphase 2 will not be eligible for retreatment. Furthermore, if a subjecthas a known neutralizing antibody, the subject will not be eligible forretreatment. However, if a non-neutralizing HAMA or HABA antibodydevelops, subjects may be retreated if they meet the eligibilitycriteria.

Post-Treatment Assessment

After completing KTE-C19 infusion and being discharged from the hospital(typically on Day 8), all subjects will be followed in thepost-treatment assessment period. Counting from day 0 (KTE-C19infusion), subjects will return to the clinic at week 2, week 4 (±3days), month 2 (±1 week), and month 3 (±1 week). Assessment can includeMMSE (mini mental status exam); PET-CT for disease assessment; physicalexam and vital signs; labs, including Chemistry Panel, CBC withdifferential, β-HCG pregnancy test (serum or urine) on all women ofchild-bearing potential, anti-KTE-C19 antibodies, lymphocyte subsets,cytokine levels, anti-CD19 CAR+ T cells, and replication-competentretrovirus (RCR) analysis; adverse/serious adverse event reporting;concomitant medications documentation; and collection of fresh tumorsample(s) for subjects who signed the optional portion of the consent.

The presence, expansion, persistence, and immunophenotype of transducedanti-CD19 CAR+ T cells will be monitored in the blood primarily by PCRanalysis, complemented by flow cytometry. Levels of serum cytokines willalso be evaluated in the blood. The following cytokines may be includedin the panel: pro-inflammatory and immune modulating cytokines IL-6,TNFα, IL-8, IL-1, IL-2, GM-CSF, IL 15, IL-17a, IFNγ, IL-12p40/p70;immune effector molecules Granzyme A, B, Perforin, sFasL; correlates ofacute phase response CRP, SAA and Chemokines MIP-1α, MIP-3α, IP-10,Eotaxin, MCP-4. As KTE-C19 comprises retroviral vector transduced Tcells, the presence of replication-competent-retrovirus (RCR) in theblood of treated patients will also be monitored.

If the subject is eligible for retreatment with KTE-C19, the last scanprior to retreatment will be considered the baseline for the purpose ofevaluating the response to retreatment.

At any time during the post-treatment assessment period, if a subjectdid not respond to treatment (i.e., CR or PR) or progresses following aresponse, the subject will proceed directly to the Month 3 visit and befollowed for disease outcomes in the long term follow-up period.

All subjects will be followed in the long-term follow-up period forsurvival and disease status, if applicable. Subjects will begin thelong-term follow-up period after they have completed the Month 3 visitof the post-treatment assessment period (whether they have responded totreatment or gone straight to the month-3 visit due to diseaseprogression). Counting from day 0 (KTE-C19 infusion), subjects willreturn to the clinic every 3 months (±2 weeks) through Month 18; every 6months (±1 month) between Month 24-Month 60; and, beginning with year 6,Month 72 (±3 months), subjects will return to the clinic 1 time annuallyup to 15 years. The following procedure will be completed at this visit:physical exam; PET-CT Scan; disease assessment; labs, including CBC withdifferential, anti-KTE-C19 antibodies, lymphocyte subsets, anti-CD19CAR+ T cells, and RCR analysis; targeted adverse/serious adverse eventreporting (for 24 months or until disease progression whichever occursfirst), including neurological, hematological, infections, autoimmunedisorders, and secondary malignancies until disease progression;targeted concomitant medication documentation (for two years afterdisease progression), including gammaglobulin, immunosuppressive drugs,anti-infective, vaccinations, and any therapy for the treatment ofprogressive diseases.

Evaluation will include baseline PET-CT scans of the neck, chest,abdomen and pelvis, along with the appropriate imaging of all othersites of disease. Subjects will have their first post KTE-C19 infusionplanned PET-CT tumor assessment 4 weeks following the KTE-C19 infusionand at regular intervals as described above.

A bone marrow aspirate and biopsy will be performed in subjects who arebeing assessed for CR. Per the revised IWG Response Criteria forMalignant Lymphoma, a bone marrow aspirate and biopsy should beperformed only when the subject had bone marrow involvement withlymphoma prior to therapy or if new abnormalities in the peripheralblood counts or blood smear cause clinical suspicion of bone marrowinvolvement with lymphoma after treatment. The bone marrow aspirate andbiopsy must show no evidence of disease by morphology, or ifindeterminate by morphology, it must be negative by immunohistochemistryto assign a CR to treatment.

Study Endpoints

Primary

The primary endpoint for Phase 1 is incidence of adverse events definedas dose-limiting toxicities (DLT). The primary endpoint for Phase 2 isthe Objective Response Rate (ORR), defined as the incidence of either acomplete response or a partial response by the revised IWG ResponseCriteria for Malignant Lymphoma as determined by the studyinvestigators. All subjects that do not meet the criteria for anobjective response by the analysis cutoff date will be considerednon-responders.

Secondary

Objective response rate among subjects in phase 1 will be summarized.Objective response rate among subjects in phase 2 will be determined perIRRC, which is defined as the incidence of either a complete response ora partial response by the revised IWG Response Criteria for MalignantLymphoma as determined by the IRRC. All subjects that do not meet thecriteria for an objective response by the analysis data cutoff date willbe considered non-responders. The duration of response (DOR) forsubjects who experience an objective response defined as the date oftheir first objective response which is subsequently confirmed todisease progression per the revised IWG Response Criteria for MalignantLymphoma or death, regardless of cause. Subjects not meeting thecriteria for progression or death by the analysis data cutoff date willbe censored at their last evaluable disease assessment date and theirresponse will be noted as ongoing.

Dose-Limiting Toxicity (DLT)

Dose-limiting toxicity is defined as the following KTE-C19-relatedevents with onset within the first 30 days following KTE-C19 infusion:

a) Grade 4 neutropenia lasting longer than 21 days from the day of celltransfer

b) Grade 4 thrombocytopenia lasting longer than 35 days from the day ofcell transfer

c) Any KTE-C19-related adverse event requiring intubation, includinggrade 4 confusion requiring intubation for airway protection isconsidered to be a DLT.

d) All other grade 3 toxicities lasting more than 3 days and all grade 4toxicities, with the exception of the following conditions which are notconsidered DLT's: i) Aphasia/dysphasia or confusion/cognitivedisturbance which resolves to grade 1 or less within 2 weeks and tobaseline within 4 weeks; ii) Fever grade 3; iii) Myelosuppression(includes bleeding in the setting of platelet count less than 50×109/Land documented bacterial infections in the setting of neutropenia),defined as lymphopenia, decreased hemoglobin, neutropenia andthrombocytopenia unless neutropenia and thrombocytopenia meet the DLTdefinition described above; iv) Immediate hypersensitivity reactionsoccurring within 2 hours of cell infusion (related to cell infusion)that are reversible to a grade 2 or less within 24 hours of celladministration with standard therapy; and v) Hypogammaglobulinemia grade3 or 4.

CRS will be graded according to a revised grading system (Lee 2014).Adverse events attributed to CRS will be mapped to the overall CRSgrading assessment for the determination of DLT.

During phase 1, approximately 6-24 subjects with DLBCL, PMBCL or TFLwill be enrolled to evaluate the safety of KTE-C19 regimens. Subjects ineach cohort will be evaluated for DLTs within the first 30 daysfollowing the completion of their respective KTE-C19 infusion. If thesubject incidence of DLT is ≦1 of 6 subjects, cohort B1 may be exploredor the study may proceed to phase 2 of the trial. This decision will bebased on overall benefit/risk and available biomarker data.

However, if 2 of the 6 enrolled subjects present with a protocol definedDLT during phase 1, the SRT may recommend enrolling 2 additional sets of3 subjects (up to 12 subjects in total) at the same dose that wasadministered in the first 6 subjects. In this scenario, progression toan additional cohort or to phase 2 of the study will proceed if ≦2 ofthe first 9 or if ≦3 of the 12 subjects present with a DLT.

If the subject incidence of DLT is >2/6, >3/9, or >4/12 subjects, otherKTE-C19 regimens may be explored in an additional 6-12 subjects (FIG.3). The same DLT rules apply as above.

Example 4

T cell products were generated by transduction of autologous lymphocyteswith a g-murine retrovirus carrying an anti-CD19 CAR construct gene,followed by expansion to achieve desired cell dose. Anti-CD19 CAR+ Tcell product characteristics were evaluated at time of harvest, or afterco-culture with CD19+ cells, by flow cytometry and multiplex cytokineanalysis of co-culture supernatants. CAR+ T cell co-culture for productcharacterization was performed with K562-CD19 cells or K562-NGFR controlcells, at an effector to target ratio of 1:1. Standard incubation timewas 18 hours. Patients with relapsed/refractory B cell malignancies wereconditioned with cyclophosphamide and fludarabine, then dosed withanti-CD19 CAR+ T cells.

Cytokine and chemokine levels were measured using EMDmillipore Luminex®xMAP® multiplex assays. Data acquisition and analysis were performedusing a Luminex 200™ instrument and xPONENT® 3.1 data analysis software.For IL-7, a human IL-7 Quantikine HS ELISA Kit (HS750) was used withsamples run neat per manufacturer's guidelines. The frequency of CAR Tcells in circulation was measured by a quantitative PCR analysis.Patients were administered a preconditioning regimen comprising of 300mg/m² cyclophosphamide on days −5 and −4, and 30 mg/m² fludarabine ondays −5, −4, and −3. Patient sera was collected prior to administrationof cyclophosphamide and fludarabine between days −12 and −5 (“pre”),immediately before administration of CAR+ T cells on day 0 (“post”) andon select days following CAR+ T cell administration up to day 18. Theserum concentrations of GF-CSF, IL-2, MCP-1, IL-6, IL-10, MCP-4, CRP,IFN gamma, Granzyme A, IL-15, IL-5, Granzyme B, IL-8, IP-10, MIP-1b,PLGF, IL-16, TARC, Eotaxin-3, sICAM-1, sVCAM-1, and SAA were measuredpre- and post-conditioning and on select days after administration ofCAR+ T cells, as shown in FIG. 6. The concentration of certain cytokineswere found to increase in patient sera post-conditioning with 300 mg/m²cyclophosphamide and 30 mg/m² fludarabine (FIGS. 7A-7I and 18A-18E). Inparticular, the concentrations of IL-15, IL-7, PLGF, CRP, and MCP-1significantly increased following conditioning with cyclophosphamide andfludarabine (FIGS. 7A-7D, 7G, 18A, and 18C-18E). Increases were alsoobserved in the concentrations of IL-5, IL-10, IP-10, and s-ICAM1 (FIGS.7E-7F, 7H-7I, and 18B). Conversely, perforin was found to decrease afterconditioning with cyclophosphamide and fludarabine (FIG. 18F). The serumconcentrations of various other analytes were observed to increase ordecrease following preconditioning, as shown in FIG. 18G. Additionalpatients were treated, and the results set forth in FIGS. 11-17. Inaddition, increased serum levels of IL-15 (FIG. 19A) and IP-10 (FIG.19B) and decreased serum levels of Perforin (FIG. 19C) followingpreconditioning were found to significantly correlate with a positiveobjective response in patients treated with CAR T cells.

Post-CAR+ T cell infusion peripheral blood lymphocytes (PBLs) and serawere evaluated by flow cytometry and multiplex cytokine analysis,respectively. Pre-infusion anti-CD19 CAR+ T cell cytokine production wascompared to a K562-NGFR negative control (FIG. 8). The concentration ofT1, T2, and immune homeostatic cytokines GM-CSF, IL-2, IFN gamma, IL-5,IL-4, and IL-13, and pro-inflammatory cytokines and chemokines TNFalpha, IL-6, Granzyme B, MIP-1b (beta), MIP-1a (alpha), and sCD137 werehigher in anti-CD19 CAR+ T cell samples relative to negative controls(FIGS. 8A-8L). In addition, engagement of the target antigen bypre-infusion product T cells lead to upregulation of receptors that canmodulate their activity, such as CD107a (alpha), 401BB, and PD-1 (FIGS.9A-9C).

Multicolor flow cytometry was carried out on a BD FACSCanto II utilizingFlowJo software for data acquisition and analysis. A shortermanufacturing process yielded CAR+ T cell products with a higherrepresentation of CD4+, naïve, and central memory T cells (FIG. 10).Post-infusion, CAR+ T cells show a diversified subset compositioncomprising mainly differentiated T cells, and some central memory ornaïve T cells (FIG. 10).

Anti-CD19 CD28zeta CAR+ T cells are clinically effective and inducedurable responses in both lymphoma and leukemia. Durable clinicalresponses can occur without long lasting CAR+ T cells in circulation,allowing normal B cell recovery. Conditioning with cyclophosphamide andfludarabine modifies the immune environment through induction ofmolecules that can favor the homeostatic expansion, activation andtrafficking of T cells. CAR+ T cell treatment results in rapid elevationand subsequent resolution of circulating cytokines and chemokines withinthree weeks after treatment.

Example 5

A study will be performed to test the safety and efficacy of treatingsubjects with a non-myeloablative conditioning regimen consisting ofdoses of cyclophosphamide greater than or equal to 300 mg/m² andfludarabine greater than or equal to 30 mg/m². The doses of theseconditioning chemotherapy agents will be used to further inducelymphocyte depletion and create a more optimal environment for expansionof KTE-C19 in vivo.

Enrolled subjects will undergo leukopheresis to obtain PBMCs for theproduction of anti-CD19 CAR+ T cells. The subjects will then receive aconditioning chemotherapy comprising 500 mg/m²/day cyclophosphamide and60 mg/m²/day fludarabine administered on day −5 to day −3. The subjectswill then receive a dose of anti-CD19 CAR+ T cells/kg by IV on day 0. Asa starting dose, subjects may receive 2×10⁶ anti-CD19 CAR+ T cells/kg(±20%), which can then be increased or decrease depending on subjectresponsiveness.

Following conditioning chemotherapy and administration of anti-CD19 CAR+T cells, the subjects will be monitored for adverse effects, serumcytokine levels, T cell counts, and disease response. The serum levelsof various cytokines, chemokines, effectors, markers of inflammation,and adhesion molecules, including, but not limited to, IL-2, IL-4, IL-5,IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, IL-21, MCP-1, IP-10, PLGF,sICAM-1, CRP, VEGF, VEGF-C, VEGF-D, sVCAM-1, MIP-1β,FGF2, IL-1b,Eotaxin, GM-CSF, IFN gamma, IL-12p40, MDC, IL-12p70, IL-13, IL-17A,MIP-1a, TNFa, TNFb, granzyme A, granzyme B, perforin, SAA, MCP-4, andTARC, will be measured before and after conditioning to determine theeffect of the conditioning chemotherapy. Serum will be collected beforeor after administration of each of cyclophosphamide, fludarabine, andanti-CD19 CAR+ T cells, and all levels will be compared to the levelsprior to conditioning chemotherapy. Disease responsiveness will becompared to each patient's the cytokine profile following conditioningto identify any correlations between disease responsiveness and thelevels of one or more cytokine following conditioning.

The occurrence of adverse effects will be closely monitored to determinethe maximum tolerable dose of cyclophosphamide and fludarabine. Adverseeffects may be medically controlled as necessary. The doses of one orboth of cyclophosphamide and fludarabine may be increased or decreasedto improve clinical efficacy and limit adverse effects. Any subjectsthat show initial partial response followed by disease progression mayreceive a second treatment at the same or a different level ofcyclophosphamide and/or fludarabine. Throughout this application,various publications are referenced in parentheses by author name anddate, or by Patent No. or Patent Publication No. Full citations forthese publications may be found at the end of the specificationimmediately preceding the claims. The disclosures of these publicationsare hereby incorporated in their entireties by reference into thisapplication in order to more fully describe the state of the art asknown to those skilled therein as of the date of the invention describedand claimed herein. However, the citation of a reference herein shouldnot be construed as an acknowledgement that such reference is prior artto the present invention. All of the various aspects, embodiments, andoptions described herein can be combined in any and all variations.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.However, the citation of a reference herein should not be construed asan acknowledgement that such reference is prior art to the presentinvention.

Having generally described this invention, a further understanding canbe obtained by reference to the examples provided herein. These examplesare for purposes of illustration only and are not intended to belimiting.

What is claimed is:
 1. A method of treating a patient having a tumorcomprising (i) administering to the patient a dose of cyclophosphamideat about 500 mg/m²/day to about 600 mg/m²/day and a dose of fludarabineat about 30 mg/m²/day and (ii) administering to the patient atherapeutically effective amount of from about 1×10⁶ to about 5×10⁶engineered CAR T cells/kg; wherein the dose of cyclophosphamide isadministered daily for three days.
 2. The method of claim 1 , whereinthe therapeutically effective amount of the engineered CAR T cells isabout 1×10⁶ or about 2×10⁶ cells/kg.
 3. A method of conditioning apatient in need of a T cell therapy comprising (i) administering to thepatient a dose of cyclophosphamide at about 500 mg/m²/day and to about600 mg/m²/day and a dose of fludarabine at about 30 mg/m²/day; and (ii)administering to the patient a therapeutically effective amount ofengineered CAR T cells; wherein the dose of cyclophosphamide isadministered daily for three days.
 4. The method of claim 3, wherein thetumor comprises non-Hodgkin's lymphoma.
 5. The method of claim 3,wherein the dose of fludarabine is administered daily for two days tofive days.
 6. The method of claim 1, wherein the dose of fludarabine isadministered daily for two days to five days.
 7. The method of claim 5,wherein the dose of fludarabine is administered daily for three days. 8.The method of claim 3, wherein the patient has diffuse large B celllymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), orfollicular lymphoma (FL).
 9. The method of claim 3, wherein theadministration of cyclophosphamide and/or fludarabine begins at leastabout five days prior to administration of the T cell therapy (day 0).10. The method of claim 1, wherein the engineered CART cells express achimeric antigen receptor that comprises an scFv antibody, wherein thescFv antibody is capable of binding a tumor antigen.
 11. The method ofclaim 3, wherein the patient has leukemia.
 12. The method of claim 10,wherein the tumor antigen is CD19.
 13. The method of claim 1 , whereinthe patient after the administration of cyclophosphamide and fludarabineexhibits an increased serum concentration, compared to the serum levelprior to the administration of cyclophosphamide and fludarabine, of ahomeostatic cytokine selected from interleukin 7 (IL-7), interleukin 15(IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma-inducedprotein 10 (IP-10), interleukin 8 (IL-8), monocyte chemotactic protein 1(MCP-1), placental growth factor (PLGF), C-reactive protein (CRP),soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascularadhesion molecule 1 (sVCAM-1), and any combination thereof.
 14. Themethod of claim 13 , wherein the homeostatic cytokine is selected fromIL-10, IL-5, IP-10, MCP-1, PLGF, CRP, sICAM-1, and any combinationthereof.
 15. The method of claim 1, wherein the patient is furthersubjected to retreatment witha dose of cyclophosphamide and a dose offludarabine.
 16. The method of claim 1 , wherein the dose of fludarabineis administered daily for two days.
 17. The method of claim 1, whereinthe dose of fludarabine is administered daily for three days.
 18. Themethod of claim 1 , wherein the dose of cyclophosphamide and the dose offludarabine are administered daily on the same day.
 19. The method ofclaim 1 , wherein the dose of cyclophosphamide is administered at leastabout one day before the dose of fludarabine.
 20. The method of claim 1, wherein the dose of fludarabine is administered at least about one daybefore the dose of cyclophosphamide.
 21. The method of claim 1, whereinthe therapeutically effective amount of T cells is about2×10⁶ engineeredCAR T cells/kg.
 22. The method of claim 3, wherein the therapeuticallyeffective amount of T cells is about 1×10⁶ or about 2×10⁶ engineered CART cells/kg.
 23. The method of claim 1 , wherein the dose ofcyclophosphamide is about500 mg/m²/day.
 24. The method of claim 1 ,wherein the dose of cyclophosphamide is about 550 mg/m²/day.
 25. Amethod of treating a patient having a tumor comprising (i) administeringto the patient a dose of cyclophosphamide at about 500 mg/m²/day and adose of fludarabine at about 30 mg/m²/day and (ii) administering to thepatient a therapeutically effective amount of engineered CAR T cells,wherein the dose of cyclophosphamide is administered daily for threedays.
 26. The method of claim 25, wherein the dose of cyclophosphamideand the dose of fludarabine are administered daily on the same day. 27.The method of claim 25, wherein the therapeutically effective amount ofengineered T cells is about 1×10⁶ or about 2×10⁶ engineered CAR Tcells/kg.
 28. A method of treating a patient having a tumor comprising(i) administering to the patient a dose of cyclophosphamide at about 600mg/m²/day and a dose of fludarabine at about 30 mg/m²/day and (ii)administering to the patient a therapeutically effective amount ofengineered CAR T cells; wherein the dose of cyclophosphamide isadministered daily for about three days; and wherein the dose offludarabine is administered daily for about three days.
 29. The methodof claim 28, wherein the T cells are CART cells.
 30. The method of claim28, wherein the therapeutically effective amount of engineered T cellsis about 1×10⁶ or about 2×10⁶ engineered CAR T cells/kg.